INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

IN RATS, THE FIRST month of postnatal life encompasses a period of rapid growth and development during which both the total body and skeletal muscle mass of neonates increase exponentially and the adult pattern of muscle contractile protein expression becomes fully established (6). During this time, growth-related signals such as thyroid hormone levels are increasing to coordinate both growth and the maturation process (13, 20, 24, 26, 30). Experimentally induced hypothyroidism has been shown to impede both general and muscle-specific growth and maturation (e.g., Refs. 16 and 25). We recently reported that the imposition of a thyroid-deficient state on neonates starting at 7 days postpartum resulted in a retardation in the growth of the heart and skeletal muscles of these animals (6). Interestingly, the growth-inhibiting effect of hypothyroidism on skeletal muscle was disproportionately greater than that for other tissues and organs. Clearly, skeletal muscle developmental processes are strongly dependent on the thyroid axis of control.

One possible mechanism for the effect of hypothyroidism on muscle growth is suggested by reports that indicate that thyroid hormone regulates components of the growth hormone (GH)-insulin-like growth factor I (IGF-I) system. For example, Nanto-Salonen et al. (24-26) have reported that hypothyroidism in neonatal rats disrupts normal developmental patterns of IGF-I and -II peptide expression and the IGF binding proteins. Consistent with this notion, we found that thyroid deficiency resulted in declining levels of circulating IGF-I at a time when euthyroid neonates were experiencing a developmentally critical "upsurge" in plasma IGF-I (6). IGF-I is thought to mediate many of the physiological effects of GH (15), and disruption of the IGF-I axis during critical developmental periods appears to have powerful effects on processes related to growth and development.

In addition to hormonal influences, skeletal muscles are known to be sensitive to changes in loading state. In adult rats, spaceflight for periods as short as 6-9 days causes atrophy of both fast- and slow-twitch skeletal muscles as well as functionally significant alterations in muscle contractile performance (e.g., Refs. 8, 10, and 11). In general, the atrophy response to unloading is more pronounced in antigravity muscles expressing greater proportions of the slow myosin heavy chain (MHC) isoform (2, 7, 11). Although the effects of muscle unloading have been studied extensively in mature mammals, the importance of muscle loading during development has not been comprehensively addressed. In one of the few such studies, Elder and McComas (14) found that chronic hindlimb unweighting in weanling rats (e.g., 18 days of age) resulted in significantly lower muscle mass and delayed myofiber phenotype development in the soleus through 18 wk of age.

In rat neonates, the period from 7 to 30 days postpartum is crucial for the development of hindlimb locomotive patterns (12). Starting at postpartum day 6-7, neonates begin pelvic weight-bearing activity, and by day 10 they have initiated true quadrapedal locomotion (12). Intermittent hindlimb unweighting of rat neonates from postpartum day 14 to 30 results in persistent disruption of gaiting patterns in adulthood (i.e., after 30 days of recovery) (31). To our knowledge, there are few or no data that address the extent to which factors known to regulate aspects of skeletal muscle growth, such as thyroid hormone, GH, and IGF-I, interact with and/or may respond to the loading conditions encountered during the time period when the neonatal rats become ambulatory.

On the basis of the foregoing information, we developed the following hypotheses. 1) The unloading of neonatal rat muscles during the developmental period from 7 days postpartum through ~20 days of age results in a significant reduction in skeletal muscle growth. 2) The same duration of unloading would have less pronounced effects on skeletal muscle growth when imposed on older neonates (e.g., ~2 wk old) whose muscles were further along the developmental cascade. 3) The imposition of a hypothyroid state would predominate over an unloading-induced reduction in muscle development. 4) Reductions in IGF-I expression mediate alterations in somatic and muscle-specific growth in response to unloading and/or the hypothyroid state.

To test these hypotheses, it was necessary to impose a chronic (i.e., continuous) unloaded state on both normal and thyroid-deficient neonatal rats. The most common method used to study the effects of unloading on freely moving limb muscles is the rat hindlimb suspension model (e.g., Ref. 4). Although this model has been applied to smaller animals such as mice and neonatal rats, the implementation of continuous hindlimb suspension in neonates is problematic due to the need for nursing at regular intervals, thereby providing a discontinuous or episodic unloading stimulus (31). Spaceflight provides the opportunity to maintain a dam and neonates together while providing continuous unloading of the limb muscles. Accordingly, we performed the studies reported herein as part of the National Aeronautics and Space Administration (NASA)/National Institutes of Health Space Life Sciences Neurolab mission, which occurred during April-May of 1998. This and the accompanying study (5) report the results of studies conducted on euthyroid and hypothyroid neonatal rats that were exposed to spaceflight for 16 days. The findings indicate that there were significant, apparently unloading-specific effects on the development of rat skeletal muscles and that hypothyroidism resulted in similar derangements that were in some cases additive with the unloading effect. In particular, both hypothyroidism and unloading significantly impacted the plasma and muscle IGF-I levels in neonatal rats.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Litter formation and experimental design. Timed pregnant dams were obtained from Taconic Farms (Germantown, NY) and housed in standard rodent cages in the vivarium at Kennedy Space Center in Florida. Shortly after birth, each litter was adjusted to n = 8 pups and matched for gender distribution. Those litters assigned to the series of integrated experiments designated as the Mammalian Development projects in the Neurolab Mission were selected on the basis of 1) the pups demonstrating normal body growth during the first 5 days of age, 2) the corresponding dams exhibiting normal water and food consumption profiles, and 3) the dams demonstrating effective maternal behaviors such as neonate retrieval. A cohort of litters from this pool was randomly assigned to the following experimental groups for this particular project: 1) 7-day euthyroid vivarium control, 2) 7-day euthyroid asynchronous ground control (AGC), 3) 7-day thyroid-deficient (TD7) vivarium control, 4) TD7-AGC, 5) 7-day euthyroid flight based, and 6) thyroid-deficient flight based. The flight groups for this component of the project were launched into space at 7 days of age.

Procedures to induce thyroid deficiency in neonatal rats. The experimental design involved the delivery of propylthiouracil (PTU) to the neonates via the dam's milk. However, technical constraints associated with the flight hardware precluded the delivery of PTU to the dam via either food or water supplies. Accordingly, PTU was administered to the dam via the implantation of mini-osmotic pumps (Alzet 2ML4), which were filled with a sterile PTU solution (56 mg/ml) under aseptic conditions. Pumps were prepared 4 h before implantation and maintained in physiological saline at 37°C to initiate flow. On the basis of nominal pump performance (2.24 ± 0.09 µl/h), this concentration of PTU delivered ~12 mg · kg¹ · day¹ to the dam (~250 g body wt) for at least 28 days. This PTU dose is approximately three times that required to completely block the conversion of L-thyroxine (T₄) to 3,5,3'-triiodothyronine (T₃) (18). The results from pilot studies indicated that this protocol rendered both the dam and nursing neonates hypothyroid (see RESULTS and Ref. 6). To validate the effectiveness of PTU delivery via the dam's milk, ground-based pilot studies were conducted that compared direct, daily PTU injections to the neonates (12 mg · kg¹ · day¹) with the effects of PTU delivery via the dam as described above.

Cage facilities for flight- and ground-based groups. The flight groups that were launched at 7 days of age were housed in research animal holding facility (RAHF) cages that were fitted into a rack located in the spacelab module, which was carried in the payload bay of the orbiter. These cages were modified to provide a "nursing" area for the dam and litter as well as a "free" area for the dam. The corresponding ground-based AGC groups were housed in modified vivarium cages of similar dimensions to the RAHF cages, whereas the ground-based vivarium controls were housed in standard vivarium cages. The older (e.g., day 14) flight group was housed in an animal enclosure module (AEM) cage designed to fit into a rack located in the middeck of the orbiter. The corresponding AGC animals were housed in cages closely approximating the configuration of the AEMs, and the ground-based vivarium controls were housed in standard vivarium cages. Two days before launch, all litters were moved to their respective flight- and ground-based cage facilities, and, 24 h before launch, the flight groups were loaded into the space shuttle. These experiments were temporally staggered so that, although the ground-based AGC and vivarium groups were processed 48 and 96 h, respectively, after that of the flight groups, the ages of each of the flight- and ground-based groups representing the "younger" and "older" neonatal groups were identical at the time of tissue procurement.

Tissue processing. Sixteen days after launch, the rats returned to Kennedy Space Center, which served as the orbiter landing site. Five hours after they landed, euthanasia (50 mg/kg Nembutal) and dissection of the rats commenced. As part of this process, blood was withdrawn from the left ventricle, the hematocrit was determined, and then the sample was centrifuged at 1,000 g for 10 min at 4°C. The resulting plasma was stored at 80°C until analyzed for IGF-I. The ventricles, soleus, plantaris, medial gastrocnemius (MG) and lateral gastrocnemius, the three vasti [intermedius (VI), medialis, and lateralis], and tibialis anterior (TA) muscles were then rapidly removed, trimmed of connective tissue, weighed, and snap frozen. All tissue and plasma samples were stored at 80°C for subsequent analyses. The tissue and plasma samples were shipped to the University of California, Irvine, on dry ice where the analytic procedures described below and in the accompanying study (5) were performed. The amount of muscle tissue, and in particular slow-twitch anti-gravity muscles, obtained from the flight groups was limited (see Table 2). Due to the apportionment of these tissues for various analyses, we chose to focus on two representative fast-twitch muscles for the complete suite of analyses presented herein. As detailed in the accompanying study (5), samples of cardiac muscle were prepared and analyzed for MHC content as a verification of the hypothyroid state.

Plasma and muscle IGF-I content. The IGF-I extraction was performed as described previously (3). Briefly, muscle samples from the TA and MG were pulverized under liquid nitrogen, and the resultant powder was transferred to tared, precooled microcentrifuge tubes for acid/ethanol extraction (9). Plasma samples were also extracted via the acid/ethanol method. The IGF-I RIA was conducted according to the manufacturer's instructions using a rat-specific RIA kit (DSL, Webster, TX).

DNA determination. The muscle DNA concentration was measured as a gross measure of continuing cellular proliferation during development. Muscle DNA was measured in whole muscle homogenates using a fluorometric assay for the DNA binding of fluorochrome bisbenzimide H-33258 (Calbiochem, San Diego, CA). Calf thymus DNA was used as a standard (22).

Statistical analysis. All values are reported as means ± SE. For each time point, treatment effects were determined by ANOVA with post hoc testing (Student-Newman-Keuls) using the Prism software package (Graphpad). Pearson's correlation analyses of relationships were performed using the Prism package. For all statistical tests, the 0.05 level of confidence was accepted for statistical significance.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

General observations. Spaceflight presented the lactating dams with a number of challenges. One of the primary concerns appears to have been the retention of the neonates in the nesting/nursing area. As a result of these stresses, a number of the younger neonates were either abandoned or destroyed by the dams in an apparent attempt to maximize the survival of the remaining offspring. During the flight, distressed or abandoned neonates were euthanized by flight crew members. After landing, NASA veterinarians examined each neonate and disqualified any apparently unhealthy animals from further analysis. As a result, the number of flight animals available for analyses was somewhat reduced (see Table 2).

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View larger version (15K): [in this window] [in a new window]   Fig. 1.   Effects of propylthiouracil (PTU) administration on the plasma L-thyroxine (T4; A) and 3,5,3'-triiodothyronine (T3; B) levels of rat neonates. In a ground-based validation study, the effectiveness of PTU delivery via the dam's milk vs. direct daily PTU injection to the neonates (see METHODS) on plasma T4 and T3 concentrations was found to be equivalent. * P < 0.05 vs. 7-day-old normal control (euthyroid) group (NC7); n = 6 per group.

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Blood hematocrit values of flight and ground control neonates following 16 days of spaceflight. * P < 0.05 vs. ground group; # P < 0.05 vs. NC7 ground; n values are given in Table 2. TD7, 7-day-old thyroid deficient (hypothyroid); NC14, 14-day-old normal control (euthyroid). Ground, age-matched ground-based controls; flight, neonates exposed to spaceflight for 16 days.

Somatic growth. Exposure to spaceflight resulted in a significant reduction in the general growth of neonatal rats (NC7 and NC14) (Fig. 3A). The imposition of a thyroid-deficient state resulted in a reduction in body mass growth that was similar in effect to spaceflight exposure and was not significantly altered further by the combination of the two interventions (Fig. 3A).

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Effects of thyroid deficiency and/or spaceflight on neonate body weight and plasma insulin-like growth factor I (IGF-I) concentration. Basal, 7-day-old neonates. A: body weight. B: plasma IGF-I concentration. * P < 0.05 vs. ground group; # P < 0.05 vs. NC7 ground; n values are given in Table 2. C: correlation between body weight and plasma IGF-I concentration with best fit line. * , Basal; , NC7 ground; , NC7 flight; , TD7 ground; , TD7 flight; , NC14 ground; , NC14 flight.

Skeletal muscle growth. The general decrease in somatic growth was also reflected in ventricular and in lower limb muscle weights from the flight vs. age-matched ground-based rats (Table 2). Because of the differences in body weight, a more meaningful presentation of these data can be obtained from the normalization of muscle weight to the individual body weight. In addition to accounting for generalized differences in somatic growth, normalized muscle weight depicts the physiologically relevant relationship between muscle mass and the potential load (body weight) imposed on limb muscles. The normalized weights of three representative muscles, in order of increasing responsiveness to spaceflight, are presented in Fig. 4. The TA is an ankle flexor that does not directly oppose gravity in normal use. Compared with the mass of the NC7 ground group, this muscle demonstrated a small but significant decline in growth as a result of exposure to spaceflight (15%) and to hypothyroid treatment (34%) and a combination of these two (41%) interventions. In the older neonates, the growth of the TA muscle was not significantly retarded (Fig. 4). The MG is a extensor of the ankle and thus would normally oppose gravity during movement. It has an MHC profile that is similar (primarily fast) to the TA (4). The MG muscles of 7-day-old rats demonstrated a 30% decrease in growth from exposure to spaceflight, whereas growth of the MG muscles from thyroid-deficient rats was depressed by 42 and 47% in the TD7 ground and TD7 flight groups, respectively (Fig. 4). The patterns of response seen in other predominantly fast MHC expressing muscles from the knee extensor and ankle extensor groups were quantitatively similar to that of the MG muscle (Table 2). The soleus is a slow-twitch MHC-expressing antigravity muscle that also contributes to ankle extension during locomotor activity and is also thought to function to maintain posture in rats. In euthyroid rats, spaceflight depressed the growth of the soleus by 50% in the 7-day-old and by 32% in the 14-day-old neonates (Fig. 4). PTU treatment resulted in a 30% decrease in soleus growth (vs. NC7 ground). The combination of thyroid deficiency and spaceflight resulted in an additional 30% decrease (e.g., total 61%) in soleus growth.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Effects of thyroid deficiency and/or spaceflight on neonate muscle mass normalized to body weight. A: muscle weight of tibialis anterior (TA), a non-weight-bearing locomotor muscle expressing primarily fast myosin heavy chain (MHC). B: muscle weight of medial gastrocnemius (MG), a weight-bearing locomotor muscle expressing primarily fast MHC. C: muscle weight of soleus, a weight-bearing postural/locomotor muscle expressing primarily slow MHC. * P < 0.05 vs. ground group; # P < 0.05 vs. NC7 ground.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Effects of thyroid deficiency and/or spaceflight on growth-related biochemical measures from neonate MG muscle. A: total muscle DNA. B: total muscle protein. C: correlation between total protein and total DNA with best fit line. * P < 0.05 vs. ground group; # P < 0.05 vs. NC7 ground.

IGF-I and muscle growth. Changes in muscle IGF-I peptide levels for the TA and MG muscles were associated with the retardation seen in muscle weights (Figs. 4 and 6). In the NC7 ground-based neonates, an apparent developmental surge in IGF-I can be seen compared with basal values (Fig. 6, A and B). This response appears to have been blunted in the flight and TD7 rats such that their IGF-I levels corresponded more closely with those of the less mature basal group. There was a significant correlation between the wet weight of the these muscles and the muscle IGF-I peptide concentration (Fig. 6, C and D). Similarly, there was a significant correlation between muscle IGF-I peptide concentration and the protein or DNA content of the MG (Fig. 7) and TA muscles (data not shown). Together, these data suggest a link between IGF-I-stimulated muscle growth (Fig. 6) and the coordination of both protein and DNA accumulation in the growing muscle.

View larger version (29K): [in this window] [in a new window]   Fig. 6.   Effects of thyroid deficiency and/or spaceflight on IGF-I peptide concentrations from neonate MG and TA muscles. A: TA IGF-I concentration. B: MG IGF-I concentration. C: correlation between muscle wet weight and muscle IGF-I peptide from all TA muscles depicted in A. D: correlation between muscle wet weight and muscle IGF-I peptide from all MG muscles depicted in B. * , Basal; , NC7 ground; , NC7 flight; , TD7 ground; , TD7 flight; , NC14 ground; , NC14 flight. * P < 0.05 vs. ground group; # P < 0.05 vs. NC7 ground.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Correlation between muscle IGF-I peptide concentration and muscle protein (A) or muscle DNA content (B) for MG muscles from all groups. * , Basal; , NC7 ground; , NC7 flight; , TD7 ground; , TD7 flight; , NC14 ground; , NC14 flight.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

In rats, the first month of postnatal life involves a remarkably dynamic period of growth and development that is regulated by complex hormonal mechanisms. To date, it has not been clear how (or if) these various growth-regulating mechanisms are impacted by environmental stimuli such as the weight-bearing activity of neonates.

A number of studies have found that thyroid hormone levels are increasing during the early postpartum time period (13, 20, 26, 30), and experimental evidence indicates that this process is important for the regulation of both general and muscle-specific growth and maturation (e.g., Refs. 16, 24, 25). However, the relationship between, or interaction of, thyroid state and weight-bearing activity during neonatal development has not been extensively characterized. It is clear that the adult pattern of muscle contractile protein expression becomes fully established by 30 days of postnatal development and that hypothyroidism during this period will result in a retardation in the growth and phenotypic development of skeletal muscles (6). Interestingly, the growth-inhibiting effect of thyroid deficiency on skeletal muscle is disproportionately greater than that for other tissues and organs.

The primary experimental groups in the present study consisted of neonatal rats that were 5-7 days of age at the onset of the experimental treatments. The period from 7 to 30 days postpartum is crucial for the development of hindlimb locomotive patterns (12). During this time, neonates will begin pelvic weight bearing (day 6-7) and initiate true quadrapedal locomotion (day 10) (12). Previous studies have found that intermittent hindlimb unweighting of rat neonates from day 14 to 30 postpartum results in persistent disruption of gaiting patterns in adulthood (31). In creating the design of these studies, it was reasoned that unweighting and/or the imposition of hypothyroidism during this time period would result in a significant perturbation of the muscle developmental program, thereby helping to elucidate the importance of these two variables in this process.

Somatic growth. In an attempt to partition the relative impact of spaceflight vs. thyroid deficiency, we have calculated the relative somatic growth deficit imposed by each treatment relative to ground-based thyroid-deficient or euthyroid controls using the data from Fig. 3A to generate Fig. 8. From this analysis, it is evident that either spaceflight or hypothyroidism resulted in an ~50% decrease in somatic growth [flight effect (NC7) vs. thyroid deficiency effect] in the younger neonates. In the thyroid-deficient neonates, the separate and combined effects of thyroid deficiency and spaceflight on somatic growth were similar, as indicated by the diminished flight effect and similar thyroid-deficient and thyroid-deficient plus flight effects. (Fig. 8). This presentation also highlights the lesser sensitivity of the older neonates (NC14) to the effects of loss of weight-bearing activity (Fig. 8, flight effect).

View larger version (20K): [in this window] [in a new window]   Fig. 8.   Relative growth deficit imposed by the separate and combined interventions of thyroid deficiency (TD) and exposure to spaceflight calculated from the body weight data presented in Fig. 3A. Flight effect was calculated as NC7 flight divided by NC7 ground, TD7 flight divided by TD7 ground, and NC14 flight divided by NC14 ground. TD effect was calculated as TD7 ground divided by NC7 ground. TD + flight effect was calculated as TD7 flight divided by NC7 ground.

Skeletal muscle growth. Compared with overall somatic growth, the growth of neonatal skeletal muscle was disproportionately impacted by the removal of weight-bearing activity during this critical developmental time period (Fig. 4). As with body weight, we have used the normalized data from Fig. 4 to apportion the relative growth deficit imposed by the separate and combined treatments of hypothyroidism and spaceflight (Fig. 9). The loss of weight-bearing activity resulted in decreased muscle growth, particularly in the younger neonates (Fig. 9, NC7 group, flight effect). In general, the mass of the non-weight-bearing TA muscles appeared to be much less sensitive to the unloading imposed by spaceflight relative to weight-bearing fast- and slow-twitch muscles such as the MG and soleus (Figs. 4 and 9). In contrast to the somatic effects of these two treatments, thyroid deficiency appeared to have a greater impact on fast muscle growth than that of spaceflight (Fig. 9, TA and MG). Whereas the interaction of thyroid deficiency and spaceflight did not result in an additional significant decrease in the mass of the TA and MG (Fig. 4, TD7 flight vs. TD7 ground), this interaction was significant in the soleus. The overall trend suggests that thyroid deficiency had the most potent effect on muscle growth, whereas the interaction of thyroid deficiency and flight may have contributed to a further decrement (Fig. 9).

View larger version (36K): [in this window] [in a new window]   Fig. 9.   Relative growth deficit imposed on TA (A), MG (B), and soleus (C) skeletal muscles by separate and combined interventions of TD and exposure to spaceflight calculated from the normalized muscle weight data presented in Fig. 4. Percent deficit was calculated by dividing normalized muscle weights as indicated on the y-axis and multiplying by 100.