INTRODUCTION

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MAMMALIAN SKELETAL MUSCLE is a phenotypically dynamic tissue capable of altering its metabolic protein profile in response to changes in physiological demand. There are many models of decreased muscle use, such as denervation, immobilization, and hindlimb unweighting, which lead to large changes in gene expression (3) and fiber size (1). A typical adaptation evident in the literature is a reduction in the levels of muscle mitochondrial proteins (16, 20). In contrast, the decreased use imposed by exposure to microgravity appears to produce an inconsistent and relatively poorly understood mitochondrial protein adaptation in skeletal muscle (1, 19). For example, 3-hydroxyacyl-CoA dehydrogenase levels were decreased in the soleus after a 7-day spaceflight (9); however, the expression of this enzyme was unaffected in vastus lateralis and vastus intermedius muscles after a 9-day exposure to microgravity (2). In addition, whereas pyruvate oxidation was unchanged by 9 days of spaceflight, the rate of palmitate oxidation was reduced (2). These data illustrate the need for further investigation into the effects of short-duration microgravity exposure on skeletal muscle metabolic enzymes. Furthermore, few measurements have been made of the mRNAs that encode metabolic enzymes (24), and no studies have compared the expression of specific mitochondrial proteins and their respective mRNA levels. In addition, much of the existing work has been related to skeletal muscle, and very little is known about the response of the heart to microgravity. During spaceflight the effects of gravity on the cardiovascular system are removed, and the blood volume that is normally pooled in the lower limbs in a 1-G environment is shifted to the thorax, thereby temporarily increasing venous return (10, 25). This shift in blood volume may cause alterations in gene expression via pathways similar to those utilized during stretch-induced cardiac hypertrophy (21). Thus the overall purpose of this study was to determine the effects of a short-duration spaceflight on cardiac and skeletal muscle mitochondria at the protein and mRNA levels of expression. This analysis should help us better understand the regulation of the expression of genes encoding mitochondrial proteins in these tissues when subjected to microgravity.

	METHODS

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Experimental animals. Adult male Sprague-Dawley rats (Taconic Farms, Germantown, NY) were exposed to microgravity for 6 days (final mass 216 ± 4 g; n = 6) aboard the Shuttle Transport System-54 mission. Similar age-matched animals (233 ± 8 g; n = 6) served as ground-based controls. All animals were fed and housed similarly, as previously described (5). Portions of the triceps brachii and heart muscles from animals subjected to microgravity were excised and snap frozen in liquid N₂ 3-9 h after the completion of the mission. Portions of the heart and triceps brachii muscles from control animals were removed on the following day over a similar time period.

Wet-to-dry mass ratios. Triceps brachii and heart fragments were pulverized to a powder at the temperature of liquid N₂. Portions of the powders were dried overnight at 70°C to a constant weight, and the ratio of wet-to-dry mass was calculated.

Enzyme analyses. Frozen triceps brachii and heart tissue powders were used to determine cytochrome c oxidase (CytOx) activity by measuring the oxidation of reduced cytochrome c (11). These tissue powders were also used to measure glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and malate dehydrogenase (MDH) enzyme activities, as previously described (18). All these enzyme activities were determined photometrically (model DU-64, Beckman) at 30°C.

mRNA measurements. Total RNA was isolated from heart and triceps tissue powders, as previously described (6). Ten micrograms of total RNA were separated on a 1% agarose minigel and transferred to a nylon membrane (Hybond-N, Amersham). RNA was fixed to the membrane with ultraviolet light, and these Northern blots were hybridized overnight with [³²P]dCTP-labeled cDNAs specific for CytOx subunits III, IV, and VIc and MDH mRNAs as well as 18S rRNA. Blots were washed initially three times for 10 min each with 2× saline sodium citrate (SSC) and 0.1% sodium dodecyl sulfate (SDS) at room temperature. All the blots were then washed for 15 min at 55°C with 0.1× SSC and 0.1% SDS and then for 15 min at 60°C with 0.1× SSC and 0.1% SDS. The levels of specific mRNAs were quantified after a 1- to 24-h exposure by using electronic autoradiography (Packard), and 18S rRNA levels were used to correct for uneven loading between lanes.

Western blotting. Proteins from triceps brachii muscle (20 µg; n = 6) and heart (15 µg; n = 6) were separated on a 15% polyacrylamide gel by using one-dimensional SDS-polyacrylamide gel electrophoresis. Proteins were electrotransferred to nitrocellulose membranes (Hybond-C, Amersham) for subsequent immunoblotting, as described previously (8, 17, 23). After blocking in 5% skim milk-1.25% horse serum, membranes were incubated with a monoclonal antibody directed against CytOx subunit IV, at a working dilution of 1:1,000 in 1% skim milk. Goat anti-mouse immunoglobulin G-conjugated alkaline phosphatase (1:750 dilution) was used as the secondary antibody. CytOx subunit IV proteins were visualized by using a color reaction, and the bands were quantified by laser densitometry.

Statistical analyses. Values are means ± SE, and statistical significance was determined by using Student's t-tests for independent samples. Differences were considered statistically significant at the 0.05 confidence level.

	RESULTS

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Wet-to-dry mass ratios. The wet-to-dry mass ratios were 4.01 ± 0.08 for control triceps brachii, 4.59 ± 0.25 for triceps brachii subjected to spaceflight, 4.10 ± 0.07 for control heart, and 4.11 ± 0.06 for heart subjected to spaceflight. There were no significant differences among these values.

Protein expression. Six days of microgravity exposure resulted in a significant 32% increase in heart mitochondrial MDH enzyme activity compared with that in control hearts. No significant effect of microgravity on heart CytOx activity or GAPDH activity was observed (Table 1). Despite the lack of change in CytOx holoenzyme activity in heart muscle, microgravity induced a 2.2 ± 0.2-fold increase (n = 6; P < 0.05) in CytOx subunit IV protein level compared with the hearts of ground-based control animals (Fig. 1). The response of skeletal muscle to microgravity differed from that of cardiac muscle. CytOx activity in the triceps muscle was significantly decreased by 41% after microgravity exposure compared with that in the triceps of control animals (Table 1). However, MDH enzyme activity in skeletal muscle remained unaffected by the microgravity treatment. In contrast, the activity of the glycolytic enzyme GAPDH was significantly elevated by 58% in the triceps muscle in response to microgravity. No effect of microgravity on CytOx subunit IV level was observed in skeletal muscle (Fig. 1).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Effects of microgravity on cytochrome c oxidase (CytOx) subunit IV protein expression in skeletal muscle and heart. Proteins from cytosolic extracts from triceps brachii (A; n = 6) and heart (B; n = 6) muscles were incubated with a monoclonal antibody directed toward CytOx subunit IV. Levels of this protein in tissues from animals subjected to spaceflight (S) and ground-based control animals (C) were quantified by using laser densitometry.

mRNA expression. Total RNA levels in the hearts of animals subjected to microgravity (1,057 ± 61 µg/g, n = 6) were not different from those in the hearts of control animals (1,025 ± 126 µg/g, n = 6). However, changes were observed in the levels of specific mRNAs. In particular, a 62% increase (P < 0.05) in MDH mRNA after microgravity exposure was observed in heart muscle (Fig. 2). The level of mRNAs encoding CytOx subunits III, IV, and VIc was modestly increased by 23-37%. In contrast to the results observed in heart muscle, the total RNA concentration in skeletal muscle was significantly decreased by 26% after the 6-day spaceflight from 916 ± 83 (n = 6) to 682 ± 42 µg/g muscle wet wt (n = 6). However, there were no corresponding reductions in the levels of the mRNAs encoding MDH or CytOx subunit III, IV, or VIc in the triceps muscle after microgravity exposure (Fig. 3).

View larger version (27K): [in this window] [in a new window]   View larger version (40K): [in this window] [in a new window]   Fig. 2.   Effect of microgravity on expression of mRNAs encoding mitochondrial proteins in heart. Total RNA (10 µg) from hearts of animals subjected to spaceflight (S) and control animals (C) were probed with cDNAs specific for mRNAs encoding CytOx subunits III, IV, and VIc and malate dehydrogenase (MDH), as well as 18S rRNA subunit. A: representative blots; B: graphical summary of all samples (n = 6), with values expressed as percentage of levels in control hearts. Arrows on left of each autoradiogram indicate levels of migration of 28S and 18S rRNAs. * P < 0.05 compared with control hearts.

View larger version (29K): [in this window] [in a new window]   View larger version (40K): [in this window] [in a new window]   Fig. 3.   Effect of microgravity on expression of mRNAs encoding mitochondrial proteins in skeletal muscle. Total RNA (10 µg) from triceps brachii of animals subjected to spaceflight (S) and control animals (C) were probed with cDNAs specific for mRNAs encoding CytOx subunits III, IV, and VIc and MDH, as well as 18S rRNA subunit. A: representative blots; B: graphical representation of all samples (n = 6), with values expressed as percentage of levels in control triceps brachii muscle. Arrows on left of each autoradiogram indicate levels of migration of 28S and 18S rRNAs.

	DISCUSSION

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The microgravity environment imposed on an organism during spaceflight provides a unique model for the study of gravitational effects on organ function. Our objective in this study was to determine the extent of the mitochondrial adaptations in cardiac and skeletal muscle after short-term microgravity exposure. Because changes in mitochondrial content are strongly correlated with performance, at least in skeletal muscle (12), our findings may have implications for muscle performance deficits observed after spaceflight (5, 7). Furthermore, characterization of these changes by using multiple enzyme measurements may help clarify the ambiguities evident in the literature with regard to the response of muscle to microgravity (1, 19). With respect to the heart, we are unaware of any study that has documented the extent of metabolic enzyme changes that occur during spaceflight in this tissue. This may provide some insight into potential changes in cardiac functional capacity during microgravity exposure. Our study was uniquely designed to evaluate not only protein measures but also coincident evaluations of changes in mRNA expression, thereby helping define precursor (i.e., mRNA)-product (i.e., protein) relationships and some underlying mechanisms responsible for the changes in protein levels observed.

Tissue-specific differences in the regulation of mitochondrial proteins have been documented previously (6, 22, 26), and our results highlight tissue-specific adaptations to short-term microgravity conditions in two different striated muscle types. Apart from the reduction in CytOx enzyme activity, gene expression in the triceps muscle remained relatively unaffected by 6 days of microgravity. Interestingly, heart muscle displayed significant increases in MDH activity and concomitant augmentations in the levels of MDH mRNA. The other mitochondrial enzyme measured, CytOx, was not significantly increased, but subunit mRNA levels tended to be higher in the hearts of animals subjected to spaceflight. These data identify a previously unrecognized cardiac muscle adaptation to microgravity, in that the heart achieves a greater oxidative capacity subsequent to spaceflight. The mechanisms responsible for inducing this adaptation remain speculative. In humans, it is known that microgravity exposure causes a redistribution of blood volume from the lower limbs to the thorax (25), thereby increasing venous return and end-diastolic volumes in the heart, at least in the early stages (4), despite a concomitant reduction in plasma volume (15). The obligatory stretching of the myocardium that accompanies this altered venous return is known to be a powerful stimulus for the induction of changes in gene expression (21). Thus alterations in the expression of mitochondrial proteins may also occur in human cardiac muscle exposed to microgravity.

Our data also illustrate the markedly different gene expression responses of mitochondrial proteins to a given physiological perturbation such as microgravity. For example, the 62% increase in MDH mRNA levels was accompanied by a 32% increase in enzyme activity in the hearts of animals subjected to spaceflight. These data suggest that in the heart the augmented levels of MDH protein may be a result of an increase in cellular mRNA levels due to altered mRNA transcription or stability matched by a somewhat lesser increase in MDH protein degradation. With regard to CytOx, a balance may have existed between the modest increase in subunit mRNAs and subunit protein degradation, leading to unaltered CytOx enzyme activity. In contrast, a qualitatively different response was observed with CytOx subunit IV, in which the small increase in mRNA level was accompanied by a much larger increase in subunit IV protein level. This suggests a decrease in subunit IV degradation rate as a result of 6 days of microgravity, and the results highlight the fact that proteins with similar organellar destinations within the same tissue may be regulated completely differently in response to a physiological perturbation.

The surprisingly large adaptations evident in the heart were not apparent in skeletal muscle, which displayed a much greater resistance to phenotypic alterations in response to microgravity exposure. For example, CytOx subunit IV mRNA and protein levels were completely unaffected by the treatment in the triceps brachii muscle. MDH mRNA and enzyme activity levels were similarly unchanged. However, because total RNA per gram of muscle was significantly reduced in this tissue, this implies that a reduction in MDH and subunit IV mRNAs did occur when expressed per gram of muscle. In order for MDH and subunit IV protein levels to remain constant (Fig. 1, Table 1), an increase in translational efficiency could have occurred, even in the absence of a change in protein degradation rate. With regard to CytOx, the catalytic properties of the holoenzyme responsible for enzyme activity reside in subunits I, II, and III (14). An increase in the translational efficiency of these catalytically relevant subunits could have been exceeded by an increase in degradation rate, resulting in the decline of enzyme activity observed in the present study. Clearly, further work is needed to corroborate the alterations in degradation rate of nuclear-encoded mitochondrial proteins, which we have interpreted to occur in response to microgravity exposure. Elevated protein degradation rates are expected to occur in view of the muscle atrophy that is observed after a 6-day spaceflight (5) as well as the enhanced protein turnover reported for nonmitochondrial proteins in skeletal muscle subject to unweighting (3).

Our data also demonstrate that the regulation of a multisubunit enzyme such as CytOx is very complex, as evident from the relatively small elevation in subunit IV mRNA, accompanied by the large increase in subunit IV protein levels in the heart, in the absence of changes in CytOx enzyme activity. It is interesting to note that the mRNAs encoding subunits III, IV, and VIc responded similarly to microgravity. This coordinated expression of mRNAs derived from the nuclear and mitochondrial genomes that code for subunits of the same holoenzyme has been previously documented under steady-state conditions (6, 11) and during conditions of altered mitochondrial biogenesis (13, 27).

The microgravity model of decreased muscle use is a valuable one that allows for the elucidation of multiple adaptations occurring in a number of different tissues during and after spaceflight. Our understanding of these adaptations will facilitate the subsequent development of appropriate countermeasures designed to eliminate or modify these changes, thereby alleviating some of the problems that occur on the return to a 1-G environment. As extensively discussed by Roy et al. (19), some of the inherent limitations in the design of experiments utilizing microgravity were evident in our study. The relatively low number of animals exposed to microgravity, the long periods of time between the return to 1 G and tissue collection (up to 9 h), and the large number of investigators involved in dividing up these tissues create significant obstacles during data collection and interpretation. Although we appreciate the logistical constraints involved in exposing animals to a microgravity environment, we believe that these design shortcomings must be addressed in future work of this kind, if possible.

In summary, the data in the present study illustrate differences in the expression of mitochondrial enzymes in heart and skeletal muscle subject to microgravity in the rat, and they reveal a surprising response in cardiac muscle subject to this condition. Further investigation into the role of protein degradation in the observed adaptations is warranted as a function of microgravity exposure time. In addition, a determination of the physiological consequences of these mitochondrial adaptations is necessary to fully understand the cardiac and skeletal muscle responses to spaceflight.