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J Appl Physiol 96: 584-596, 2004. First published October 17, 2003; doi:10.1152/japplphysiol.00724.2003
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Adaptations in metabolic capacity of rat soleus after paralysis

Jeffrey S. Otis,1 Roland R. Roy,2 V. Reggie Edgerton,2,3 and Robert J. Talmadge4

1Muscle Function Laboratory, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061; 2Brain Research Institute and 3Department of Physiological Science, University of California, Los Angeles 90095; and 4Biological Sciences, California State Polytechnic University, Pomona, California 91768

Submitted 14 July 2003 ; accepted in final form 10 October 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
To determine whether long-term reductions in neuromuscular activity result in alterations in metabolic capacity, the activities of oxidative, i.e., succinate dehydrogenase (SDH) and citrate synthase (CS), and glycolytic, i.e., {alpha}-glycerophosphate dehydrogenase (GPD), enzyme markers were quantified in rat soleus muscles 1, 3, and 6 mo after a complete spinal cord transection (ST). In addition, the proportional content of lactate dehydrogenase (LDH) isozymes was used as a marker for oxidative and glycolytic capacities. The myosin heavy chain (MHC) isoform content of a fiber served as a marker of phenotype. In general, MHC isoforms shifted from MHC1 toward MHC2, particularly MHC2x, after ST. Mean SDH and CS activities were higher in ST than control at all time points. The elevated SDH and CS activities were indicative of an enhanced oxidative capacity. GPD activities were higher in ST than control rats at all time points. The increase in activity of SDH was larger than GPD. Thus the GPD-to-SDH (glycolytic-to-oxidative) ratio was decreased after ST. Compared with controls, total LDH activity increased transiently, and the LDH isozyme profile shifted from LDH-1 toward LDH-5, indicative of an enhanced glycolytic capacity. Combined, these results indicate that 1) the metabolic capacities of soleus fibers were not compromised, but the interrelationships among oxidative and glycolytic capacity and MHC content were apparently dissociated after ST; 2) enhancements in oxidative and glycolytic enzyme activities are not mutually exclusive; and 3) chronic reductions in skeletal muscle activity do not necessarily result in a reduced oxidative capacity.

citrate synthase; {alpha}-glycerophosphate dehydrogenase; lactate dehydrogenase; myosin heavy chain isoforms; succinate dehydrogenase

IT IS GENERALLY ACCEPTED THAT the number and frequency of electrical impulses received by a muscle fiber play significant roles in modulating the quantities of metabolic enzymes and types of contractile proteins that are expressed by that muscle fiber (41, 49, 52). In turn, these protein systems regulate force generation, maximal velocity of shortening, fatigability, and metabolic substrate utilization. For example, chronic low-frequency electrical stimulation of rabbit fast muscles results in a near complete transition to a slow phenotype, including an elevated oxidative capacity (41). Other models of increased neuromuscular activity (e.g., endurance exercise training) also result in elevations in oxidative capacity as measured by the levels of marker enzymes, e.g., succinate dehydrogenase (SDH) or citrate synthase (CS), or by the percentage of fiber volume occupied by mitochondria (3, 29). This is typically accompanied by a net reduction in glycolytic capacity, e.g., reductions in phosphofructokinase activity (35). These adaptations support the theory that chronically increased neuromuscular activity induces a net change in fiber-type composition from fast glycolytic toward slow oxidative (41).

Therefore, a widely held view is that a reduction in neuromuscular activity results in a shift toward a faster fiber phenotype, including a reduction in oxidative capacity and an elevation in glycolytic capacity. Numerous studies have documented that reduced activity results in a shift toward faster phenotypic protein isoforms. For example, after spinal cord injury (SCI), paralyzed muscles atrophy (5, 10, 44, 51), and the expression of myosin heavy chain (MHC), isoforms transitions from slow (MHC1) to faster (MHC2a, MHC2x, and MHC2b) isoforms (18, 19, 44, 49, 50, 53-55).

Several studies have documented that short-term hindlimb unloading, as occurs with spaceflight or ground-based models of spaceflight, such as hindlimb suspension, results in decreased or unchanged oxidative and increased glycolytic capacities in the hindlimb muscles (12, 22, 24, 36, 39, 60). These data have been interpreted to mean that a decrease in neuromuscular activity induces a reduction in oxidative and an elevation in glycolytic capacities. However, the idea that hindlimb unloading equates to a reduction in activity is controversial. For example, we have demonstrated that, although there is an immediate reduction in soleus electromyographic activity associated with hindlimb suspension, the activity levels return to normal levels within 10 days after unloading (2). Thus the muscular responses to chronic hindlimb unloading may be different from those induced by models that result in sustained reductions in activation.

The present study was undertaken to determine the long-term adaptations in the oxidative and glycolytic capacities after spinal cord transection (ST), a model that results in sustained reductions in the electrical activation of the affected muscles (1). Specifically, we investigated whether the oxidative and glycolytic capacities were compromised in a normally slow rodent muscle, i.e., the soleus, after ST. Measurements were made at time points up to 6 mo post-ST, a duration representing ~15% of the animals' life expectancy (30). Relative to the transitions in MHC isoform expression that occur after ST, the three time points chosen represent an early (1 mo), middle (3 mo), and late (6 mo) transitional stage (55). Based on the reported increase in fatigability and shift from slower to faster MHC isoforms after ST (53, 55, 56), it was hypothesized that the oxidative capacity of the soleus fibers would be reduced and the glycolytic capacity elevated after ST.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Experimental animals. Female Sprague-Dawley rats (~170 g and ~45 days of age) were assigned to six groups (n = 5/group): 1) 1-mo control; 2) 1-mo post-ST (1-Mo ST); 3) 3-mo control; 4) 3-mo post-ST (3-Mo ST); 5) 6-mo control; and 6) 6-mo post-ST (6-Mo ST). Animals assigned to the ST groups were subjected to a complete transection of the spinal cord at a midthoracic level. Details of the surgical procedures and postsurgical care have been described in detail elsewhere (45, 53, 55, 56). The rats used in this study were young adult females, which show a higher degree of viability after ST than older or male rats. In addition, rats of this age appear to have a mature complement of oxidative and glycolytic enzymes (4). All procedures were performed in accordance with the US Government Principles for the Utilization and Care of Vertebrate Animals and were approved by the University of California, Los Angeles, Virginia Polytechnic Institute, and California State Polytechnic University at Pomona animal care committees.

At each time point studied, the rats were terminated by lethal injection of pentobarbital (100 mg/kg body wt ip), and the soleus muscles were removed, weighed, and quickly frozen in melting isopentane, chilled with liquid nitrogen. The muscles were stored at -70°C.

Quantitative histochemistry of individual muscle fibers. The maximal activity of SDH (EC 1.3.5.1 [EC] ) served as a marker for the oxidative capacity, and the maximal activity of {alpha}-glycerophosphate dehydrogenase (GPD) served as a marker for the glycolytic capacity of individual muscle fibers (40, 42, 43). As stated by Rivero et al. (43), it is not known whether this histochemical method stains for the activity of the NAD-dependent GPD (EC 1.1.1.8 [EC] ) or the mitochondrial FAD-dependent GPD (EC 1.1.99.5 [EC] ). Neither GPD enzyme is directly involved in the glycolytic pathway; however, both are directly involved in the transfer of NADH from glycolysis in the cytosol into FADH2 in the mitochondria of skeletal muscle. Therefore, GPD activity correlates with the activities of other glycolytic enzymes (34). A minimum of 20 serial cross sections were cut from the midbelly of each soleus in a cryostat (Microm HM505) at -20°C and adhered to coverslips. Five of the sections were used for SDH analysis. Three 10-µm-thick sections were stained in a medium containing (in mM) 100 potassium phosphate buffer (pH 7.6), 1.0 methoxy phenazine methosulfate, 0.75 NaN3, 1.5 nitroblue tetrazolium, 5 ethelyene diamine tetraacetic acid, and 10 succinic acid for 8 min at 25°C, as described by Blanco et al. (7). Two sections were incubated in the same medium without substrate (succinic acid) and served as reaction controls. Similar procedures were used for analysis of GPD activity. Three 14-µm-thick sections were stained in a medium containing (in mM) 100 potassium phosphate buffer (pH 7.4), 0.02 methoxy phenazine methosulfate, 1 NaN3, 1.2 nitroblue tetrazolium, and 6.3 {alpha}-glycerophosphate for 11 min at 37°C, and two sections were incubated in the same medium without substrate ({alpha}-glycerophosphate) and served as reaction controls (37).

Images of ~30 stained fibers from the central portion of the midbelly of each muscle were digitized into computer files within 2-3 h after the histochemical reactions by using a Nikon Eclipse E400 microscope, Cohu high-performance charge-coupled device camera, and computer-controlled image analysis software (Scion Image version 1.62). Images of the fibers were saved as digitized images at 256 gray levels. The gray levels were converted to optical density (OD) units by using a calibrated set of OD filters. The digitized images of the fibers within the selected region were traced manually and analyzed for fiber cross-sectional area (CSA) and the average OD for each histochemical reaction. The average fiber OD for each histochemical reaction was determined as the average OD for all pixels within the traced fiber from the sections incubated with substrate minus the average OD for all pixels of the same fiber from the sections incubated without substrate. As stated by Blanco et al. (7), the mean OD measurement reflects the specific enzymatic activity per unit fiber volume (i.e., enzyme concentration), because each pixel represents a standardized fiber volume (pixel length x width x section thickness). The integrated enzymatic activities (ISDH and IGPD) were calculated as the product of specific enzymatic activity (mean OD for a given fiber) and CSA of that fiber. Because the volume of the fiber that was measured for staining intensity equals the product of section thickness (which is held constant) and fiber CSA, the integrated enzyme activities reflect the enzyme content per fiber.

Immunohistochemistry. Immunohistochemical analysis of MHC isoform content in individual fibers was performed on the 10 remaining serial cryostat sections, as described by Talmadge et al. (53, 55). Briefly, 10-µm-thick sections were stained using a series of monoclonal antibodies (MAb) specific for rat MHC isoforms, and the fibers were classified for MHC content according to their individual patterns of MAb reactivity (see Table 1 for MAb specificity and Table 2 for MHC-based fiber-type MAb reactivity). Two of the sections were used to control for nonspecific binding of the secondary antibodies (i.e., the IgG- and IgM-specific secondary antibodies) and were subjected to the same staining protocol as the other sections, but without primary antibody. For enzymatic analyses, fibers containing MHC1 plus any type 2 MHC (MHC2a, MHC2x, or MHC2b) were collapsed into a single group and defined as MHC1/2 hybrid fibers.


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  		Table 1. Myosin heavy chain monoclonal antibody specificity

 

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  		Table 2. Immunohistochemical identification of fiber MHC isoform content

 

Biochemical assays. CS (EC 4.1.3.7 [EC] ) activities were determined according to Srere (48). Briefly, portions (encompassing the entire muscle cross section) of the same frozen muscles used for histochemical analyses were minced with scissors and homogenized by hand on ice using ground-glass homogenizers in CS homogenization buffer, which consisted of 20 mM imidazole (pH 7.0), 50 mM NaF, 2 mM ethelyene diamine tetraacetic acid, 20% glycerol, and 0.1 mM phenylmethylsulfonyl fluoride. The crude homogenates were centrifuged at 4°C for 25 min at 14,000 g. The supernatant was retained for CS activity measurements. CS activity was assessed in a final volume of 1 ml in a reaction medium containing 100 mM Tris (pH 8.1), 0.65 mM oxaloacetic acid, 0.15 mM acetyl-CoA, and 0.1 mM DTNB and monitored at 25°C for 1 min using a Bio-Rad SmartSpec 3000 spectrophotometer at 412-nm wavelength to detect the formation of reaction product, i.e., 2-nitro-5-thio-benzoic acid (mercaptide ion). Protein concentration of the supernatants was assessed according to the Bradford technique (8) using bovine serum albumin as a protein standard. All CS activities are expressed as nanomoles of product formed per milligram protein per minute.

Lactate dehydrogenase (LDH; EC 1.1.1.27 [EC] ) isozyme content was assessed by native, agarose gel electrophoresis followed by staining for LDH activity. Portions (encompassing the entire muscle cross section) of the same muscles used for histochemical and CS assays were homogenized in extraction buffer containing 30 mM NaCl, 3 mM MgCl2, 0.02% Nonidet P-40, and 20 mM Tris (pH 8.0) and centrifuged at 4°C at 600 g for 5 min. The supernatant was saved for LDH isozymes electrophoresis. Supernatant protein was determined according to Bradford (8). Forty micrograms of supernatant protein were loaded per lane. The horizontal gels consisted of 1.3% agarose, 25 mM Tris, and 192 mM glycine, pH 8.6. LDH isozymes were stained for 40 min in the dark at 37°C in a reaction medium consisting of 50 mM Tris (pH 8.0), 23 mM l-lactic acid, 1 mM MgCl2, 0.2 mg/ml NAD+, 0.2 mg/ml nitro-blue tetrazolium, and 0.05 mg/ml phenazine methosulfate. The bands were scanned using an Alpha Innotech AlphaImager 2000 Gel Imaging workstation. For each time point, all control and ST samples were run on the same gel. Because some gel-to-gel staining variability may exist, the LDH isozyme data are expressed as 1) the proportion of each isozyme band relative to the sum of all bands and 2) the absolute intensity of each band relative to the same band in controls. Total LDH activity was calculated as the sum of all bands per lane and is expressed relative to control.

Statistics. All data are presented as means ± SE. Data were analyzed by using the SigmaStat 2.03 statistical program. A two-way analysis of variance, group (ST or control) by time (1, 3, or 6 mo), was used for overall comparisons. For single-fiber data, individual comparisons between groups were analyzed via the Kruskal-Wallis test, because some data were not normally distributed. For all other comparisons, Student-Newman-Keuls post hoc tests were used to compare control vs. ST at that particular time point. Significance was accepted at P < 0.05. Because the biochemical, histochemical, and immunohistochemical analyses revealed no time-dependent changes in the control soleus, i.e., there were no significant effects of age for the control groups, except for fiber size (which is shown in Fig. 2), the data from control animals at all time points were combined into one group for simplicity of presentation.



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  		Fig. 2. Mean (±SE) fiber cross-sectional area (CSA) measurements for all fibers combined, i.e., regardless of MHC-based fiber type (A), MHC1 fibers (B), hybrid fibers (C), MHC2a fibers (D), MHC2a/2x fibers (E), and MHC2x fibers (F) from 1-, 3-, and 6-mo control and 1-, 3-, and 6-Mo ST rats. No. of fibers analyzed per group was 149 from 1-mo control, 153 from 3-mo control, 155 from 6-mo control, 153 from 1-Mo ST, 154 from 3-Mo ST, and 155 from 6-Mo ST. *Significantly different from control, P <= 0.05. {dagger}Significantly different from 1-Mo ST, P <= 0.05.

 


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Fiber MHC composition. See Fig. 1. In control rats (regardless of the time point), ~95 and ~1% of the fibers were identified as pure MHC1 and pure MHC2a, respectively. Approximately 2% of the fibers were identified as MHC1/2 and ~2% as MHC2a/2x. One month after ST, the proportion of MHC1 fibers decreased, such that only ~49% contained only MHC1, and the proportion of MHC1/2 hybrid fibers (i.e., MHC1/2a, MHC1/2a/2x, and MHC1/2x combined) was increased to ~40%. The 1-Mo ST rats also had ~3% MHC2a, ~5% MHC2x, and ~3% MHC2a/2x fibers. Three months after ST, only ~9% of the fibers were pure MHC1 fibers, and the proportion of hybrid 1/2 fibers increased to ~71%. The 3-Mo ST rats also had ~5, ~3, and ~12%, MHC2a, MHC2a/2x, and MHC2x fibers, respectively. At 6-mo post-ST, the proportion of pure MHC1 fibers remained at ~6%. In addition, the proportion of hybrid 1/2 fibers was 0%, and the proportions of MHC2a, MHC2a/2x, and MHC2x fibers were increased to ~13, ~30, and ~51%, respectively.



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  		Fig. 1. Myosin heavy chain (MHC)-based fiber-type proportions in the soleus of control, 1-mo spinal cord transected (1-Mo ST), 3-mo ST (3-Mo ST), and 6-mo ST (6-Mo ST) rats. Values are means ± SE; n = 15 control rats (457 fibers) and 5 ST rats per time point (153, 154, and 155 fibers for the 1-, 3-, and 6-Mo ST rats, respectively). I, MHC1; IIa, MHC2a; IIx, MHC2x. The hybrid MHC-I/IIa, I/IIa/IIx, and I/IIx fibers were collapsed into a single group, identified as MHC-I/II, for subsequent analyses. Significantly different from *control, {dagger}1-Mo ST, and {ddagger}3-Mo ST: P <= 0.05.

 

Fiber CSA. See Fig. 2. The mean fiber CSA of the control rats increased between the 1- and 3-mo time points, i.e., from ~2.5 to ~4.5 mo of age. No further growth occurred between the 3- and 6-mo time points, i.e., from ~4.5 to ~7.5 mo of age (Fig. 2A). Compared with age-matched controls, the overall fiber CSA was decreased by 43, 56, and 45% after 1, 3, and 6 mo of ST, respectively. In addition, the CSA was larger in 6-Mo ST than 1-Mo ST rats. The mean CSA of pure MHC1 fibers was ~54% of control after 1 mo of ST, but returned to ~73% of control CSA by 6 mo post-ST (Fig. 2B). The CSAs of MHC1/2 hybrid and pure MHC2a fibers were similar to control at 1-mo post-ST, but smaller than control at subsequent time points (Fig. 2, C and D). Fibers containing some MHC2x, i.e., MHC2a/2x and MHC2x fibers, were observed almost exclusively in ST rats. There was a trend (P > 0.05) for these fibers to increase in size between 1 and 3 mo after ST and then to plateau (Fig. 2, E and F).

Enzyme activities and interrelationships: all fibers combined. See Fig. 3. The mean SDH activity of all fiber types combined was higher than control at all time points post-ST and higher at 6 than 1 and 3 mo post-ST (Fig. 3A). Mean ISDH, a measure of the total SDH enzyme content in a fiber, was similar to control at 1 and 3 mo post-ST and greater than control 6 mo post-ST (Fig. 3B). In addition, the mean ISDH values were higher at 6 than 1 and 3 mo post-ST. Mean GPD activity was elevated at all time points post-ST compared with control (Fig. 3C). This value peaked at 3 mo (2.7-fold increase) and was lower (26% reduction) at 6 than at 3 mo post-ST. Compared with control, the mean IGPD, a measure of the total glycolytic power of the fiber, was similar at 1 mo and higher at 3 and 6 mo post-ST compared with control (Fig. 3D). In addition, both the GPD and IGPD values were lower at 6 than 3 mo post-ST (Fig. 3, C and D). The GPD-to-SDH ratio (GPD/SDH) was lower than control at 1 and 6 mo post-ST and lower at 6 than 1 mo post-ST (Fig. 3E).



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  		Fig. 3. Mean (±SE) enzyme activities for all fibers combined (i.e., regardless of MHC-based fiber type) from control and 1-, 3-, and 6-Mo ST rats. A: succinate dehydrogenase (SDH) activity. B: integrated SDH (ISDH) activity. C: {alpha}-glycerophosphate dehydrogenase (GPD) activity. D: integrated GPD (IGPD) activity. E: the GPD-to-SDH activity ratio (GPD/SDH). OD, optical density. Nos. of rats and fibers are the same as in Fig. 1. *Significantly different from control, P <= 0.05. {dagger}Significantly different from 1-Mo ST, P <= 0.05.

 

Enzyme activities and interrelationships of specific fiber types. See Figs. 4, 5, 6, 7, 8. Pure MHC1 fibers showed similar adaptations for all enzymatic measures following ST, except IGPD, as all fibers combined (compare Figs. 3 and 4). MHC1/2 fibers, which were not present in 6-Mo ST rats, showed consistent elevations in SDH, GPD, and IGPD activities post-ST (Fig. 5, A, C, and D), whereas mean ISDH activity was only transiently increased at 1 mo post-ST (Fig. 5B). In contrast to all fibers combined, GPD/SDH increased in the MHC1/2 hybrid fibers (Fig. 5E). The MHC2a fibers also showed similar trends in enzymatic adaptation as all fibers combined (compare Figs. 3 and 6). MHC2a/2x fibers showed no change in SDH activity after ST (Fig. 7A), although ISDH was increased in 3- and 6-Mo ST rats relative to 1-Mo ST rats (Fig. 7B). Mean GPD activity was increased at 1 and 3 mo post-ST, but decreased to control levels 6 mo post-ST (Fig. 7C). In contrast, mean IGPD activity showed a continuous increase up to 6 mo post-ST (Fig. 7D). Compared with control, GPD/SDH of the MHC2a/2x fibers was increased at 1 and 3 mo post-ST (Fig. 7E). Direct comparisons to control for the MHC2x fibers observed in ST rats cannot be made because the control rat soleus did not contain this fiber type. The MHC2x fibers showed a reduction in SDH activity with time after ST (Fig. 8A). Unexpectedly, the levels of SDH activity (~30-50 OD/min x 10-3) in these fibers were higher than in control MHC1 fibers (~15 OD/min x 10-3) (compare Figs. 4A and 8A). The MHC2x fibers showed a decrease in GPD activity with time after ST, and these values (~7-14 OD/min x 10-3) were higher than in control MHC1 fibers (~3-4 OD/min x 10-3) (compare Figs. 4C and 8C). The GPD/SDH of the MHC2x fibers was higher at 3 than 1 or 6 mo post-ST (Fig. 7E).



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  		Fig. 4. Mean (±SE) enzyme activities for MHC1 fibers from control and 1-, 3-, and 6-Mo ST rats. A: SDH activity. B: ISDH activity. C: GPD activity. D: IGPD activity. E: GPD/SDH. No. of fibers analyzed per group was 436 from control, 75 from 1-Mo ST, 13 from 3-Mo ST, and 9 from 6-Mo ST. *Significantly different from control, P <= 0.05. {dagger}Significantly different from 1-Mo ST, P <= 0.05.

 


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  		Fig. 5. Mean (±SE) enzyme activities for hybrid MHC1/2 fibers from control, 1-Mo ST, and 3-Mo ST rats. No hybrid MHC1/2 fibers were observed in 6-Mo ST rat soleus muscles. A: SDH activity. B: ISDH activity. C: GPD activity. D: IGPD activity. E: GPD/SDH. No. of fibers analyzed per group was 9 from control, 60 from 1-Mo ST, and 109 from 3-Mo ST. *Significantly different from control, P <= 0.05. {dagger}Significantly different from 1-Mo ST, P <= 0.05.

 


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  		Fig. 6. Mean (±SE) enzyme activities for MHC2a fibers from control and 1-, 3-, and 6-Mo ST rats. A: SDH activity. B: ISDH activity. C: GPD activity. D: IGPD activity. E: GPD/SDH. No. of fibers analyzed per group was 5 from control, 5 from 1-Mo ST, 8 from 3-Mo ST, and 21 from 6-Mo ST. *Significantly different from control, P <= 0.05; {dagger}Significantly different from 1-Mo ST, P <= 0.05.

 


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  		Fig. 7. Mean (±SE) enzyme activities for hybrid MHC2a/2x fibers from control and 1-, 3-, and 6-Mo ST rats. A: SDH activity. B: ISDH activity. C: GPD activity. D: IGPD activity. E: GPD/SDH. No. of fibers analyzed per group was 8 from control, 5 from 1-Mo ST, 5 from 3-Mo ST, and 46 from 6-Mo ST. *Significantly different from control, P <= 0.05. {dagger}Significantly different from 1-Mo ST, P <= 0.05.

 


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  		Fig. 8. Mean (±SE) enzyme activities for MHC2x fibers from 1-, 3-, and 6-Mo ST rats. No MHC2x fibers were observed in control rat soleus muscles. A: SDH activity. B: ISDH activity. C: GPD activity. D: IGPD activity. E: GPD/SDH. No. of fibers analyzed per group was 8 from 1-Mo ST, 19 from 3-Mo ST, and 79 from 6-Mo ST. *Significantly different from control, P <= 0.05. {dagger}Significantly different from 1-Mo ST, P <= 0.05.

 

To corroborate the adaptations in SDH, CS activity was used as a whole muscle marker for oxidative capacity. CS activity was elevated compared with control at all three time points after ST (Fig. 9). The CS activities of the control rat soleus were similar to previously reported values (35, 47). To corroborate the adaptations in {alpha}-GPD, LDH isozymes were measured as markers of glycolytic and oxidative capacity. In the soleus muscles of the ST rats, there was a shift in the LDH isozyme profile from predominantly LDH-1, normally found in slow-oxidative muscles, toward LDH-5, normally found in fast-glycolytic muscles (Fig. 10). In addition, total LDH activity was increased only transiently in ST rat soleus (Fig. 10C).



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  		Fig. 9. Citrate synthase (CS) activities for soleus muscles from control (solid bars) and ST rats (open bars) at 1, 3, and 6 mo after ST. The activities are expressed as nmol product formed per mg protein in the supernatant fraction per min. Values are means ± SE. *Significantly different from corresponding control, P <= 0.05.

 


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  		Fig. 10. Lactate dehydrogenase (LDH) isozyme content (A and B) and total LDH activity (C) of soleus muscles from control and 1-, 3-, and 6-Mo ST rats. A: LDH isozyme content expressed as percentage of the sum of all LDH isozymes per muscle. B: LDH isozyme content expressed relative to the control levels (the horizontal dashed line demarks the control levels). C: total LDH content expressed relative to the control levels (the horizontal dashed line demarks the control levels). Values are means ± SE. *Significantly different from corresponding control, P <= 0.05.

 


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
The primary findings of the present study were that neither oxidative nor glycolytic capacities of rat soleus fibers were decreased after paralysis induced by a complete transection of the spinal cord at a midthoracic level. In fact, despite a slow-to-fast adaptation in the MHC isoform content, oxidative capacity was increased. The data do not support our hypothesis that oxidative capacity would be reduced after ST. The results suggest that long-term reductions in neuromuscular activity do not necessarily result in a reduced oxidative capacity of a normally slow oxidative rodent muscle.

Adaptations in oxidative and glycolytic capacities and MHC isoforms after ST. Based on quantitative histochemical assays of individual muscle fibers, SDH activity is maintained in the cat soleus muscle after ST (28, 33) or spinal cord isolation (SI; a model of complete inactivity) (25). However, the adaptations in phenotypic proteins related to contraction, i.e., MHC, are somewhat modest in slow muscles of cats after ST and SI (54). In fact, only ~50% of the fibers in the normally homogeneously slow cat soleus muscle contain fast-MHC isoforms after 6 mo of paralysis (54). In contrast, the rat soleus shows a more rapid and complete transition from slow- to fast-MHC isoforms after ST or SI (27, 31, 53, 55). Thus it was expected that the rat soleus would undergo a transition from a slow-oxidative to a fast-glycolytic profile after a prolonged period of ST. In contrast, our results suggest that the transition is toward a fast-oxidative glycolytic phenotype. These data are similar to those from cats, as well as a mixed muscle (i.e., the vastus lateralis) after SCI in humans (11). GPD and LDH activities increase, at least transiently, in fast and slow muscles in rats, cats, and humans after ST, SI, and SCI (present study; Refs. 11, 25, 28, 33, 34). Thus there appears to be some consistency in that both the oxidative (SDH and CS) and glycolytic capacities (GPD and LDH) are either maintained or elevated in slow- or mixed-fiber-type muscles after chronic reductions in neuromuscular activity associated with SCI.

Overall CS, SDH, and ISDH levels progressively increased with time after ST. In contrast, GPD and IGPD levels, although higher than control, were significantly decreased in most fiber types at the later time points (from 3 to 6 mo) relative to 1 mo after ST. In addition, total LDH activity in ST rats was increased only transiently above control levels. These adaptations in metabolic properties are reflected in the decreased GPD/SDH (glycolytic-to-oxidative ratio). In contrast, Hoffmann et al. (28) reported an increased reliance on glycolytic metabolism in the soleus and medial gastrocnemius muscles 6 mo after ST in kittens transected at 2 wk of age. The present results, however, suggest that this increased reliance on glycolytic metabolism may be transient. Therefore, as the MHC profile of the paralyzed soleus transitioned from a predominantly slow muscle and stabilized as that of a predominantly fast muscle 6 mo after ST, the glycolytic, but not the oxidative, capacity transitioned toward levels normally observed in fast fibers.

In general, the adaptations in the enzymatic activities of all MHC-based fiber types were similar, although not identical. Most fiber types displayed an increased oxidative capacity and a transient increase in glycolytic capacity after ST. Because most MHC-based fiber types showed significant changes in both their oxidative and glycolytic capacities, a dissociation between substrate metabolism and MHC isoform expression apparently occurred over time. Furthermore, as occurs following chronic low-frequency stimulation or functional overload of a muscle (17, 41), for a given MHC-based fiber type, the metabolic adaptations appear to precede the adaptations in proteins associated with speed-related mechanical properties, e.g., MHC. Because previous studies have demonstrated that perturbations in substrate utilization by either creatine phosphate depletion or hypobaric hypoxia induce adaptations in other phenotypic properties of muscle fibers (6, 32, 38), it is possible that metabolic adaptations may signal or enforce signals that induce phenotypic adaptations of proteins associated with mechanical properties, as suggested previously by Moerland et al. (38) and Dunn and Michel (17).

It is likely that multiple cellular mechanisms contribute to the elevated oxidative and glycolytic capacities observed in the soleus fibers after ST. First, because the quantitative histochemical method for assessing SDH activity measures the mean specific activity (staining intensity) throughout the fiber CSA, any reduction in fiber CSA with a commensurate maintenance of total SDH activity within the fiber would concentrate the SDH activity (and mitochondrial volume) in a smaller CSA (and fiber volume) and result in increased SDH activity. Because we did observe a reduction in fiber CSA, it is likely that this "concentrating effect" contributed to the elevated SDH activity of the individual fibers and indicates that not all protein systems of a fiber are negatively influenced by the atrophic process. For instance, myofibrillar proteins may be reduced, whereas mitochondria and their components apparently are not. Second, the "concentrating effect" does not completely explain the enhanced SDH activity in the soleus fibers of the ST rats. The ISDH measure reflects the total SDH content per fiber (mean specific SDH activity x CSA of the fiber). If the "concentrating effect" were the sole mechanism involved in producing the elevated SDH activity, then the ISDH value would remain relatively constant. However, this value also increased after ST, suggesting that actual mitochondrial biogenesis and/or SDH protein expression was enhanced in the soleus, despite the loss of contractile proteins. In contrast, 11 wk after ST, the fast rat vastus lateralis shows a reduction in SDH activity (26). The signaling mechanisms responsible for the elevated SDH and ISDH after ST in rat slow muscle are presently unknown.

Relationship to fatigability. Typically, muscles with a high proportion of oxidative fibers fatigue more slowly than muscles with a low proportion of oxidative fibers when subjected to repetitive stimulation of equal relative intensity. However, it is clear that a dissociation between the oxidative profile and the fatigability of a muscle can occur under some conditions. For example, Simoneau et al. (46) demonstrated that, after prolonged periods (>14 days) of chronic low-frequency stimulation, rabbit and rat fast muscles reach a plateau in fatigue resistance, despite a continued increase in oxidative capacity, as measured by CS activity. Castro et al. (11) recently demonstrated that human vastus lateralis muscle fibers have elevated SDH activities, as measured by quantitative histochemistry, despite an increased fatigability of the quadriceps complex following short-term SCI. This issue is supported by data from animals that have had their hindlimbs unloaded by actual spaceflight. For instance, spaceflight of 7-14 days results in nonsignificant changes in rat soleus fiber SDH activity (36, 39). However, reductions in rat soleus fatigue resistance occurred following a 6-day spaceflight (9). In contrast, hindlimb suspension, a ground-based model for spaceflight, results in no change in either soleus fiber SDH (39) or fatigue resistance (58). Whole body suspension, another ground-based model for spaceflight, also results in little change in rat soleus fatigue resistance (21). In contrast, chronic immobilization of the soleus in a shortened position resulted in a more rapid loss of soleus force output during repeated contractions compared with controls (59). The differences in the response of the rat soleus to these varying models of "reduced activity or load-bearing state" highlight the idea that each of these models is physiologically distinct.

The present study shows an increase in fiber oxidative capacity, despite a reported decrease in fatigue resistance in the rat soleus 3 and 6 mo post-ST (56). In total, these studies suggest that the oxidative properties of a muscle are not always directly related to the fatigability of the muscle. This suggests that some other point within the neuromuscular unit becomes the "weak link" with respect to fatigability. Several studies have suggested that the excitation-contraction coupling process becomes impaired with fatigue (20, 23, 57), and anatomic and functional changes in the excitation-contraction coupling apparatus occur after paralysis (14-16, 50). Future studies should directly address the involvement of the excitation-contraction coupling process in the increased fatigability observed after ST and SCI. It is important to mention that none of the studies that show a dissociation between fatigability and oxidative capacity of a muscle have measured the actual flux of substrates through the oxidative pathway and the resultant ATP synthesized. Consequently, it is also possible that other aspects of the oxidative metabolism-based synthesis of ATP may be impaired.

There is a prevailing idea in the literature that slow-MHC1 fibers have higher oxidative enzyme activities than fast-MHC2 fibers and that this greater oxidative capacity is responsible for the higher resistance to fatigue observed among muscles with high than low proportions of MHC1 fibers. In contrast, studies that have quantified the oxidative capacities of single rodent fibers identified according to their MHC isoform composition show that MHC2a and MHC2x fibers have higher SDH activities than MHC1 fibers (42, 43). These studies documented that the SDH activities of single fibers from the deep region of the rat medial gastrocnemius were highest in MHC2a fibers, followed in decreasing order by MHC2x, MHC1, and MHC2b fibers. Similarly, in a study of a large number of rat skeletal muscles, Delp and Duan (13) have demonstrated that CS activity is more highly correlated with the proportion of MHC2a in a muscle than MHC1. Our present data are in agreement: we observed a higher oxidative capacity (SDH activity) in fast-MHC2a and -MHC2x fibers than MHC1 fibers. In total, our data are consistent with a general transformation from a slow- to a fast-fiber phenotype, including an upregulation of oxidative capacity to resemble that found in fast fibers of control rat soleus.

Finally, it is interesting to note that the ST-induced adaptations from predominantly MHC1 to predominantly MHC2x fibers did not require the transition through the MHC2a phenotype. This is evidenced by the high proportion of MHC1/2x hybrid fibers present at 1 and 3 mo after ST. Thus the fiber transitions were not restricted to the nearest neighbor transitional scheme (41), as we have previously demonstrated (53, 55).

Conclusions. The present results demonstrate that the oxidative and glycolytic capacities of rat soleus muscle fibers are not compromised after ST. This is consistent with previous data from humans after SCI and supports the hypothesis that increased fatigability observed after chronic reductions in neuromuscular activity are not due to a compromised capacity for ATP synthesis (11). These results suggest that 1) under certain circumstances skeletal muscles may undergo increases in both oxidative and glycolytic enzyme activities simultaneously, and 2) chronic reductions in muscle activation are not necessarily associated with reductions in oxidative capacity.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
This work was funded in part by National Institutes of Health (NIH) Grant S06 GM-53933 and American Paralysis Association Grant TA1-9402-1 (to R. J. Talmadge) and NIH Grant NS-16333 (to V. R. Edgerton and R. R. Roy).


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
The authors thank Nicole D. Garcia and Jung A. Kim for excellent technical assistance. We thank Dr. S. Schiaffino (University of Padova, Italy) for providing some of the monoclonal antibodies.

Present address of J. S. Otis: Department of Pharmacology, Emory University School of Medicine, 5001 Rollins Research Center, Atlanta, GA 30322-3090.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. J. Talmadge, Biological Sciences, California State Polytechnic Univ., Pomona, CA 91768 (E-mail: rjtalmadge{at}csupomona.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 

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