INTRODUCTION

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CITRATE SYNTHASE (CS) is one of the key regulatory enzymes in the energy-generating metabolic pathway that catalyzes the condensation of oxaloacetate and acetyl coenzyme A to form citrate in the tricarboxylic acid cycle. It has been extensively used as a metabolic marker in assessing oxidative and respiratory capacity (22). More than 30 years ago, Holloszy and his colleagues (8) reported that endurance exercise training increases the oxidative enzyme activities in skeletal muscle. After that, numerous studies were conducted to examine the effect of exercise training on muscle oxidative capacity and metabolic characteristics (7, 15, 20, 22, 29). It is generally recognized that the skeletal muscle oxidative capacity and the activities of mitochondrial enzymes are elevated by endurance exercise training.

Transient changes in skeletal muscle mitochondrial enzyme activities have been investigated after a single bout of exercise in both animals (5, 9, 21) and humans (14, 27). Data from the human studies suggested that CS activities elevate in response to acute exercise, whereas conflicting findings in the CS activity changes after an acute bout of exercise were reported in the rat studies (5, 9, 12, 13, 26). Unaltered or even decreased CS activities have been reported in skeletal muscles shortly after a single bout of exercise. Several studies have been conducted to determine the influence of exercise training in the mitochondrial enzyme adaptation in cardiac muscles (2, 10, 11, 18, 19). Although the majority of previous studies suggest that the training-induced alteration of mitochondrial enzymes is not noticeable in cardiac muscle (1, 2, 17, 18, 30), a few discrepant results regarding the changes were reported (10). Moreover, among the studies that have been conducted to examine the exercise-induced response of CS in cardiac muscle, very few have provided data regarding the transcriptional, translational, and/or posttranslational processes. Thus, whereas exercise-associated changes in CS have been well studied, the molecular mechanism for CS adaptation to endurance training in cardiac muscle is not well understood.

Adaptive elevation in the mitochondrial protein content by exercise training is suggested to be due to an increase in the rate of protein synthesis rather than a decrease in the rate of protein degradation (3, 24, 25). This raises the possibility that the modulation of mitochondrial enzyme expression in response to exercise training is regulated by pretranslational, translational, and/or posttranslational control mechanisms. In fact, an increase in any particular mRNA concentration may signal an increased synthesis of that particular protein (28). Because the underlying mechanism(s) for the changes in CS activity induced by endurance training and acute exercise has not been completely elucidated, the purpose of this study was to examine the acute and chronic responses of CS gene expression and enzymatic activity in rat skeletal and cardiac muscles after 8-wk endurance treadmill training. We hypothesized that the responses of cardiac and skeletal muscles to acute exercise and endurance training as measured at 1 and 48 h after the last training session of the endurance training would have similar CS gene expression and activity.

	METHODS

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Animals. Male adult (3 mo of age) Sprague-Dawley rats (Harlan, Indianapolis, IN) were studied. Animals were housed in pathogen-free conditions, two per cage at 20-22°C with a reversed 12:12-h light-dark cycle, and fed rat chow and water ad libitum throughout the study period.

Training protocol. Sixteen animals ran on a level motorized rodent treadmill (Columbus Instruments, Columbus, OH) 5 days weekly for a period of 8 wk. During the first 4 wk, the speed of the treadmill and duration of the training sessions were gradually increased from a speed of 10 m/min for 10 min to a running speed of 28 m/min for 55 min by the end of the fourth week. During weeks 5 through 8, a 5-min warm-up session at a speed of 20 m/min was followed by the 55-min training session at a speed of 28 m/min. During the training sessions, mild electrical shock stimulation was applied, if necessary, to maintain the running motivation.

RT-PCR analysis of CS mRNA. Total RNA was isolated from rat soleus and cardiac muscles with use of TriReagent (Molecular Research Center, Cincinnati, OH), which is based on the guanidine thiocyanate method. Frozen muscles were mechanically homogenized on ice in 1 ml of ice-cold TriReagent. RNA was solubilized in RNase-free H₂O and quantitated in duplicate by measuring the absorbance at 260 nm. Two micrograms of RNA were reverse transcribed with decamer primers and Superscript II reverse transcriptase (RT) in a total volume of 20 µl according to standard methods (Invitrogen, Life Technologies, Bethesda, MD). Control RT reactions were done in which the RT enzyme was omitted. The control RT reactions were PCR amplified to ensure that DNA did not contaminate the RNA. One microliter of cDNA was then amplified by PCR using 100 ng of each forward and reverse CS primers, 250 µM deoxyribonucleotide triphosphate, 1× PCR buffer, and 2 units Taq DNA polymerase (Sigma Chemical, St. Louis, MO) in a final volume of 50 µl. PCR were performed by using a programmed thermocycler (Biometra, Göttingen, Germany). A primer pair was designed against CS (forward 5'-CCGTGCTCATGGACTTGGGCCTT- 3'; reverse 5'-CCCCTGGCCCAACGTAGATGCTC-3'). The primers were designed with an annealing temperature at 59.9°C. All PCR products were verified by restriction digestion and by sequencing. Preliminary experiments were conducted with CS primers to ensure that the number of cycles represented a linear portion for the PCR optical density curve and within the exponential phase of the PCR amplification for the muscle samples. The cDNA from all muscle samples was amplified simultaneously by using aliquots from the same PCR mixture. After the PCR amplification, 15 µl of each reaction was electrophoresed on 1.5% agarose gels, stained with ethidium bromide. Then images were captured and the signals were given in arbitrary units as optical density × band area by use of the Kodak one-dimensional image analysis system (Eastman Kodak, Rochester, NY). The size (number of base pairs) of each of the bands corresponded to the size of the processed CS mRNA. In the present study, ribosomal 18S were used as internal controls, and all RT-PCR signals were normalized to the 18S signal of the corresponding RT product (Ambion, Austin, TX). This eliminated the measurement error from uneven sample loading and provided a semiquantitative measure of the relative changes in CS gene expression.

CS activity measurement. Ventricle and skeletal muscles (~20 mg) were homogenized on ice in 0.1 M Tris buffer containing 0.1% Triton X-100, pH 8.35. CS activity was determined spectrophotometrically according to the method of Srere (23). The homogenates were frozen under liquid nitrogen and thawed four times to disrupt the mitochondria to expose the CS. The assay system contained in a total volume of 200 µl:100 mM Tris buffer (pH 8.35), 5 mM 5,5-dithiobis(2-nitrobenzoate) (DTNB), 22.5 mM acetyl-CoA, 25 mM oxaloacetate (OAA), and 4 µl of homogenate of muscle. The principle of the assay was to initiate the reaction of acetyl-CoA with OAA and link the release of free CoA-SH to a colorimetric reagent, DTNB (acetyl-CoA + OAA + H₂O  citrate + CoA-SH, then CoA-SH + DTNB  mercaptide ion). The rate change in color was monitored at wavelength of 405 nm at 15-s intervals for a period of 3 min by using a Dynex MRX plate reader controlled through personal computer software (Revelation, Dynatech Laboratories). All measurements were performed in duplicate, in the same setting at 20-22°C. The solubilized protein extracts of the homogenates were quantified in duplicate by using bicinchoninic acid reagents (Pierce, Rockford, IL) and bovine serum albumin standards. The CS activity was then normalized to the total protein content and was reported in as nanomoles per milligram protein per minute.

Statistical analyses. Results are presented as means ± SE. One-way ANOVA was used to examine differences across groups. Post hoc differences between the means from experimental groups were determined via Tukey's tests. Significance was set at P < 0.05.

	RESULTS

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Characterization of animals. At the start of the study, animals in all groups had similar body weights (210-240 g). All animals assigned to the T₁ and T₄₈ groups successfully completed the treadmill training protocol. After the training, the animals were ~5 mo old. Trained animals (both T₁ and T₄₈) weighed less than the sedentary control animals at the time of death (T₁ and T₄₈ vs. C: 406 ± 9.9 and 386 ± 10.2 vs. 449 ± 12.9 g, P < 0.05).

CS gene expression. CS mRNA content was analyzed by RT-PCR in soleus and cardiac ventricle muscles of C, T₁, and T₄₈ animals. Representative ethidium bromide-stained gels for the CS mRNA are shown in Figs. 1 and 2. The soleus CS mRNA levels were 21 and 18% greater in T₁ and T₄₈ animals, respectively, compared with sedentary C animals (P < 0.05) (Fig. 1). Similarly, cardiac CS mRNA levels were 23 and 17% higher in muscle isolated from T₁ and T₄₈ animals, respectively, compared with those isolated from C animals (P < 0.05) (Fig. 2).

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Representative RT-PCR results from soleus muscles isolated from sedentary control (C) rats and trained rats killed 1 or 48 h after last training (T1 and T48, respectively). PCR products were visualized with ethidium bromide staining and were analyzed by densitometry given in signal product area × optical density (OD). Expression of the citrate synthase (CS) gene is normalized to the ribosomal 18S signal from the same RT product. Normalized data are presented as means ± SE. Data were run in duplicate on different gels for both genes. *P < 0.05, significantly different from C animals.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Representative RT-PCR results from cardiac muscles isolated from C, T1, and T48 animals. PCR products were visualized with ethidium bromide staining and were analyzed by densitometry given in signal product area × OD. Expression of the CS gene is normalized to the ribosomal 18S signal from the same RT product. Normalized data are presented as means ± SE. Data were run in duplicate on different gels for both genes. *P < 0.05, significantly different from C animals.

CS activity. CS activities were measured biochemically in soleus and cardiac ventricle muscles of C, T₁, and T₄₈ animals. CS activities increased by 42 and 25% in soleus muscles from T₁ and T₄₈ animals, respectively, relative to activities in C animals (T₁ vs. C: 469 ± 18.9 vs. 330 ± 6.6; T₄₈ vs. C: 412 ± 10.9 vs. 330 ± 6.6 nmol · mg protein¹ · min¹, P < 0.01). The soleus muscle from T₁ had a 14% higher level of CS activity compared with the muscle from T₄₈ (T₁ vs. T₄₈: 469 ± 18.9 vs. 412 ± 10.9 nmol · mg protein¹ · min¹, P < 0.05) (Fig. 3). However, cardiac CS activities were not significantly changed with exercise training in either T₁ or T₄₈ animals compared with C animals (Fig. 4).

View larger version (8K): [in this window] [in a new window]   Fig. 3.   CS activities for soleus muscles in C, T1, and T48 animals. CS activity is normalized to the total protein content of the sample used in the assay. Normalized data are presented as means ± SE. *P < 0.05, significantly different from C animals. #P < 0.05, significantly different from T48 animals.

View larger version (7K): [in this window] [in a new window]   Fig. 4.   CS activities for cardiac muscles in C, T1, and T48 animals. CS activity is normalized to the total protein content of the sample used in the assay. Normalized data are presented as means ± SE.

	DISCUSSION

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This study demonstrated that 8 wk of endurance treadmill training in rats increases the CS activity in skeletal soleus muscle but not in cardiac ventricle muscle. Here we provide evidence that suggests that exercise has an acute effect on the training-induced increase in CS activity. These data are in agreement with human studies showing that the acute CS activity in skeletal muscle is increased after a single bout of exercise (14, 27). Although different acute and chronic adaptive responses of CS activities to endurance training were found between soleus and cardiac ventricle muscles, both of the muscle types demonstrated a similar modulation of CS mRNA level in the animals 1 and 48 h after the final session of exercise training. However, the data in the present study do not show whether there are different molecular adaptations (translational and/or posttranslational mechanism) of CS adaptations to exercise training in rat soleus and cardiac muscles or whether the heart already has maximal levels of CS, so that training cannot further increase these levels. Further studies are needed to determine whether either mechanism can explain the different CS adaptive responses to training reported in skeletal and cardiac muscles.

Chronic response of CS activity to training. Chronic effects of exercise training (8-12 wk) on skeletal muscles CS activity have been extensively examined in rats (1, 6, 20) and humans (4, 14). In the present study, we have found that soleus muscle from the trained rats 48 h after the last training session had a 25% higher level of CS activity compared with the sedentary rats (T₄₈ vs. C: 412 ± 10.9 vs. 330 ± 6.6 nmol · mg protein¹ · min¹, P < 0.01). This is consistent with the previous findings, and the data fall into published ranges of CS activity elevation from similar duration and intensity of exercise training (1, 14, 20, 30). Moreover, we reported that the CS activities in cardiac ventricle muscle were unchanged after 8-wk endurance treadmill training in both groups of 1 and 48 h after the final session of exercise training, a finding that agrees with the majority of the previous findings. There is little evidence in the literature reporting that exercise training promotes an increase in CS activity in rat cardiac muscle (10). Our findings are consistent with previously published data indicating that CS activity is not altered after endurance training in rat cardiac muscle (1, 2, 17, 18, 30). It was suggested that myocardium has sufficient preexisting oxidative capacity to supply the energy requirement during exercise.

Acute response of CS activity to exercise. Although it appears clear that endurance exercise training promotes a chronic effect of CS activity elevation in skeletal muscle, results on the acute effects of endurance exercise on CS activity are less clear. Tonkonogi and co-workers (27) investigated the acute effect of a single bout of prolonged cycling on the oxidative function in human skeletal muscle. They observed that skeletal muscle CS activity was increased by 20% measured in millimoles per kilogram dry weight per minute (increased by 12% measured in related to protein content) immediately after an acute bout of exhaustive cycling in ~75% peak O₂ uptake. A recent study conducted by Leek and colleagues (14) reported that an acute bout of exercise lasting for 30 min significantly increases the CS activity 1 h after the cessation of exercise in the skeletal muscle from both sedentary and trained humans. They have shown that there is a 50% increase in the CS activity in 1 h after the acute exercise and an 18% increase in the CS activity in the skeletal muscle after 8 wk of training. In summary, the findings from previous human studies consistently demonstrated that an acute bout of exercise upregulates the CS activity in skeletal muscle. There is evidence supporting the conclusion that a single bout of exercise (at least 30 min) exerts an increase in the skeletal muscle CS activity that would overshadow the CS activity elevation resulting from chronic endurance training in humans (14). In contrast to the human studies, several previous rat studies have reported unchanged or even decreased CS activities immediately after a single bout of exercise (9, 12, 26). In the present study, we have shown that acute exercise significantly increases the soleus CS activity by 42% (measured in relation to protein content) 1 h after cessation of prolonged exercise in trained rats compared with the sedentary control rats. In addition, we found a 25% increase in the soleus CS activity of the trained rats 48 h after the last training session compared with the sedentary control rats. The greater level of soleus CS activity in T₁ animals compared with T₄₈ animals suggests that an acute effect of exercise on the training-induced elevation in CS activity exists in rats; therefore, it is important to choose appropriate points after the last bout of exercise if the chronic effects of exercise are to be fully evaluated. Choosing a sampling time too close to the last exercise session will probably overestimate the effect of chronic exercise on CS levels. Collectively, the present findings indicate that the elevation in CS activity after an acute bout of exercise occurs at 1 h after the cessation of exercise.

CS gene expression after exercise training. Our findings demonstrated that the CS mRNA levels in soleus and cardiac ventricle muscles from both the T₁ and T₄₈ animals were elevated by ~20% compared with sedentary control animals. Previous findings suggested that upregulation of CS gene transcription occurs within 3 h after a single bout of exercise and the elevated CS mRNA level is still present 24 h after exercise (16). Our present data have added to the previous findings of others by showing that the CS mRNA level in soleus muscle is elevated by 21% when comparing rats 1 h after the last training session with sedentary control rats. We have also reported that there is an 18% increase in soleus CS mRNA level in rats 48 h after the last training session compared with sedentary control rats. We have provided novel evidence indicating that there is an upregulation of CS gene transcription in rat soleus muscle occurring at both 1 and 48 h after the last session of endurance training. We have shown that the soleus CS activity is significantly increased by 42% when comparing rats 1 h after the last training session with sedentary control rats. This could not be completely explained by the 21% upregulation of CS gene transcription in soleus. However, we cannot rule out the possibility that CS mRNA content may have also changed at the time points other than 1 h after the last training session. Further studies are needed to determine whether modification on the active site of the existing CS proteins may be important in the regulation of CS enzymatic activity. We speculate that this may be a reasonable explanation for our findings because it was demonstrated that covalent enzyme modification may occur by phosphorylation and dephosphorylation or oxidation and reduction, which is related to the increased muscle CS activity (9).