The mammalian respiratory pump is a multi-muscle system. The actions of many ventilatory muscles provide a means by which gas exchange can be maintained under a wide range of metabolic demands (e.g., rest to heavy exercise). Although the diaphragm is the primary muscle of ventilation, it is clear that other muscles are also activated during quiet breathing(8). Indeed, it is known that expiration is an active process during eupnea and is associated with phasic activation of various muscle groups in several species (7,8).
The rat has long been used as an animal model to study respiratory muscle physiology. Although the diaphragm has been studied extensively(9,19-23), limited information exists concerning the myosin ATPase activity, myofibrillar protein isoforms, and the bioenergetic capacity of other ventilatory muscles in the rat. This is significant because the diversity of skeletal muscle depends upon the interaction of myofibrillar protein isoforms and the enzymes involved in energy metabolism (16,18). These parameters have not been systematically analyzed within a single investigation in rat ventilatory muscles and thus form the rationale for this study.
Therefore, the objective of this study was to characterize bioenergetic enzyme activities and myosin phenotype of both inspiratory (e.g., diaphragm) and expiratory (e.g., rectus abdominis) muscles in healthy adult rats. We postulated that a clear metabolic distinction should exist between primary muscles involved in ventilation and those muscles that play only a minor role. Specifically, we hypothesized that the primary inspiratory muscles (i.e., costal and crural diaphragm) would exhibit a higher oxidative enzyme activity and a higher percentage of slow myosin isoforms compared with less active ventilatory muscles (i.e., sternomastoid, intercostals, rectus abdominis). To test this hypothesis, we measured the myosin phenotype and activities of oxidative (citrate synthase) and glycolytic (lactate dehydrogenase) enzymes in rat inspiratory and expiratory muscles.
Animals. These experiments were approved by the Institutional Animal Care and Use Committee and followed the principles established by both the American College of Sports Medicine and the American Physiological Society for animal research. To standardize housing and environmental factors, eight healthy female Sprague-Dawley rats were housed for 3 months in 22 × 42 cm cages and provided rat chow and water ad libitum.
Tissue removal. At the time of tissue removal, animals were approximately 7 months old. Animals were anesthetized with pentobarbital sodium (50 mg·kg-1 i.p.). When a surgical plane of anesthesia was achieved, samples of costal diaphragm (CO-D), crural diaphragm (CR-D), sternomastoid (SM), parasternal intercostals (PI), internal intercostals (II), external intercostals (EI), rectus abdominis (RA), and plantaris (PL) were removed for subsequent biochemical analysis. To compare the biochemical properties between muscles that assist in respiration and muscles that function solely in locomotion, the plantaris muscle was selected to be representative of a phasic, mixed-fiber, locomotor muscle.
In an effort to standardize muscle sampling procedure, all muscles were removed by one experienced investigator. Diaphragm segments were obtained from the right and left anterior costal as well as the crural region. The PI, EI, and II were exposed by reflection of the overlying tissue and muscle samples removed between the second and sixth intercostal spaces; muscles from both the right and left sides of the sternum were removed. The PI samples were restricted to intercostal muscle located within 0.5 cm of the sternum. The EI and II muscles were carefully separated by the investigator using the visual assistance of a dissection microscope. The RA was exposed by a midsagittal incision of the abdominal wall skin; the incision began immediately inferior to the xiphoid process and extended to the inguinal region. Finally, the SM was exposed by an incision of the overlying skin. All samples were quickly removed, frozen in liquid nitrogen, and stored at -80 °C until assay.
Analysis of bioenergetic enzyme activities. After thawing, muscle samples were quickly dissected free of fat and tendon before homogenization. Muscle were homogenized in cold 100 mM phosphate buffer with 0.05% bovine serum albumin (1:100 wt·vol-1; pH = 7.4). Homogenization was achieved by 10 passes of the homogenate in a tight-fitting glass-on-glass homogenizer. Homogenates were then centrifuged (3 °C) for 10 min at 400× g. The resulting supernatant was decanted and assayed for citrate synthase (CS: E.C. 188.8.131.52) activity using the procedures described by Srere (26), and lactate dehydrogenase (LDH: E.C. 1.1.27) activity was determined using the technique of Bergmeyer et al.(3). CS and LDH enzyme activities were interpreted as relative measures of metabolic capacities of the Krebs cycle (CS) and glycolysis (LDH), respectively.
In our hands the coefficients of variation (CV) for CS and LDH assays were≈3 and 5%, respectively. All enzyme assays were performed in duplicate at 25 °C and samples from all animals were assayed on the same day to reduce interassay variation. Total protein in the homogenate was assayed using the technique described by Bradford (5).
Analysis of myofibrillar ATPase activity. Muscle samples assayed to determine myofibrillar ATPase (mATPase) activity were homogenized and assayed using the technique described by Caiozzo et al.(6). mATPase activity was measured as an index of the muscle's ability to hydrolyze ATP, and mATPase activity has been shown to be highly correlated with muscle maximal shortening velocity(1). All assays were performed in duplicate at 25 °C and samples from all animals were assayed on the same day to reduce interassay variation. Total protein in the homogenate was assayed using the technique described by Bradford (5). In our laboratory the CV for the mATPase assay is 5%.
Metabolic differentiation paradigm. To determine the bioenergetic metabolic properties of respiratory muscles, we used the metabolic differentiation paradigm proposed by Pette et al.(2,17). This model assumes that metabolic differentiation in striated muscle is related to the relative capacities of aerobic and anaerobic bioenergetic pathways and that assessment of glycolytic and oxidative enzyme activities are relative measures of the capacities of these pathways. Further, this model employs the ratio of enzyme activities of aerobic and anaerobic metabolic pathways to distinguish among muscles with different metabolic characteristics. We chose CS and LDH as our markers of aerobic and anaerobic pathways, respectively. CS activity, a Krebs cycle enzyme, is considered a standard marker of the relative oxidative capacity of a tissue since it is highly correlated with mitochondrial respiratory capacity(12,15). LDH is not considered the rate limiting enzyme in glycolysis, but LDH activity has been shown to be higher in tissues with a high glycolytic capacity (15). Hence, muscles with high oxidative capacity will have low LDH/CS ratios, whereas muscles with low oxidative capacity and high glycolytic capacities will have high LDH/CS ratios.
It is important to note that although the LDH/CS activity ratio accurately characterizes the metabolic type of skeletal muscle, it does not reflect the absolute metabolic potential. Therefore, the LDH/CS activity ratio can only be used for comparative purposes in similar types of tissues. For example, the LDH/CS activity ratio in smooth muscle might suggest characteristics of an aerobic (red) skeletal muscle although its overall oxidative enzyme activities are considerably lower than that of red skeletal muscle(2,17). In contrast, comparison of the LDH/CS activity ratio between different skeletal muscles provides an accurate index of the “metabolic type” of muscle (2).
Determination of myosin isoforms. Myosin is a hexameric myofibrillar protein and consists of two myosin heavy chains (MHC) and four light chains (MLC). We measured both MHC and MLC composition to classify the myofibrillar protein phenotype of the muscles studied. In adult rat skeletal muscle, four MHC isoforms have been identified: 1) type I MHC; 2) type IIa MHC; 3) type IId MHC: and 4) type IIb MHC. Each MHC is associated with both an alkali and a regulatory MLC. Three alkali MLC isoforms exist in adult rats(13,18,27): 1) type 1 slow(1s); 2) type 1 fast (1f); and 3) type 3 fast (3f). Two regulatory MLC isoforms exist: 1) type 2 slow (2s); and 2) type 2 fast (2f).
MHC composition was assessed using SDS-polyacrylamide gel electrophoresis procedures described by Talmadge and Roy (28). Briefly, after determination of protein content, a sample of denatured myofibrillar protein was loaded onto 16-cm long vertical gels (8% polyacrylamide; glycerol 30% v·v-1) and electrophoresed for 20 h at 8 °C. Gel lanes containing both molecular weight standards and myofibrillar samples from a slow muscle (soleus) and mixed fiber muscle (costal diaphragm of Sprague Dawley rat) were run simultaneously with experimental samples as standards. Further, the migration pattern of the slow MHC isoform was confirmed by using monoclonal antibodies against the slow type I MHC isoform. For identification purposes the gels were stained with Coomassie blue R-250 and destained by diffusion in a methanol/glacial acetic acid solution. The relative concentrations of myosin heavy chain isoforms were determined by scanning the gels using a computerized densitometric image analysis system.
MLC composition was assessed using SDS-polyacrylamide gel electrophoresis procedures described by Schluter and Fitts (25). Briefly, a sample of denatured myofibrillar protein was loaded onto 8-cm long vertical gels (12% polyacrylamide; glycerol 30% v·v-1) and electrophoresed for ≈1 h at 8 °C. Gel lanes containing both molecular weight standards and isolated preparations of myosin light chains (Sigma Chemical Co., St. Louis, MO) were run simultaneously with experimental samples as standards. Gels were stained with Coomassie blue R-250, destained, and the relative concentrations of MLC isoforms were determined as previously described for MHC's.
Statistical analysis. Data were analyzed using ANOVA. Significance was established at P < 0.05. An a priori decision was made to use a Bonferoni (t-test) comparison between groups to permit multiple comparisons. Also, to examine the relationships between enzyme activities and myosin isoforms, simple correlation coefficients were computed for all measured variables.
Muscle enzyme activities. Specific activities (means ± SEM) of CS and LDH for all muscles are contained inFigures 1 and 2, respectively. The highest mitochondrial enzyme activity (CS) was found in the diaphragm. Specifically, CS activity was significantly higher in the CO-D and CR-D compared with that in all other respiratory muscles as well as in the plantaris. Further, CS activity was approximately 20% higher in the CO-D compared with that in the CR-D. The lowest respiratory muscle CS activity was found in the RA; all other muscles had significantly higher CS activity compared with that in the RA. CS activity did not differ between the SM, PS, II, EI, and PL muscles.
LDH activity in the CO-D and CR-D was lower compared with that in all other muscles studied, whereas the highest LDH activity was found in the SM(Fig. 2). No differences existed in LDH activity between the PS, II, EI, RA, and PL muscles.
Figure 3 contains the ratio of LDH to CS activity for each of the muscles studied. According to the metabolic differentiation paradigm (2), the ratio of LDH to CS activity provides an index of the relationship between the glycolytic capacity and the oxidative capacity of the tissue. Note that the numerical values of these enzyme ratios are dimensionless and thus represent purely a magnitude for comparison. Examining the LDH/CS ratio makes it possible to discriminate the muscles into three general groups: group 1) CO-D and CR-D (low LDH/CS ratios); group 2) SM, PS, II, EI, PL (moderate LDH/CS ratios); and group 3) RA (high LDH/CS ratio). In regard to these three groups, the LDH/CS ratios are significantly lower in both the CO-D and CR-D compared with those in all other muscles. The low ratio of LDH/CS in the diaphragm is indicative of a high oxidative capacity and relatively low glycolytic capacity. In contrast, the high LDH/CS activity ratio in the RA suggests that this muscle possesses a relatively high glycolytic capacity and a relatively low oxidative capacity. The fact that the LDH/CS activity ratios for the SM, PS,II,EI, and PL muscles fall between the diaphragm and the RA suggests that these muscles have lower oxidative capacities than the diaphragm but higher oxidative capacities than the RA.
Figure 4 contains the mean (± SEM) mATPase activity for all muscles studied. Myofibrillar ATPase activity was significantly lower in the CO-D and CR-D compared with that in all other muscles. No differences in mATPase activity existed between the SM, PS, II, EI, RA, and P1 muscles.
Muscle myosin isoforms. The MHC isoform profile of the rat respiratory and locomotor muscles studied are contained inTable 1. Compared with all other muscles, the CO-D and CR-D contained the highest percent of type I and lowest percent of type IIb MHC isoform. Significant differences existed between the CO-D and CR-D in all four MHC isoforms. In general, no differences existed in MHC isoforms between the SM, PS, II, EI, and RA muscles.
Table 2 contains the MLC isoform profile of the rat respiratory and locomotor muscles studied. Compared with all other muscles, the CO-D and CR-D contained the highest percentage of type I slow MLC and lowest percentage of type 1f MLC. Interstingly, the CR-D contained the highest percentage of 3f MLC, whereas the CO-D contained the lowest percentage of type 3f MLC compared with other respiratory muscles.
MLCs were also analyzed by analysis of ratios of specific alkali MLCs (seeTable 3). A comparison of MLC ratios has been used by other investigators to examine both the binding pattern of MHC's with specific alkali MLCs and to assess the functional significance of the relative concentrations of specific alkali MLCs (9,29). In the present study, two MLC ratios were used to classify muscles regarding their relative amount of slow and fast alkali MLC composition. First, the ratio of MLC 3 f/(1s + 1f + 3f) was examined as an index of the relative amount of MLC 3f. This is significant because the maximal shortening velocity of single muscle fibers increases with increasing amounts of MLC 3f(9,27). Second, the ratio of MLC 1s/(1s + 1f + 3f) was used as an index of the relative amount of slow alkali MLC.
Interestingly, the highest 3 f/(1s + 1f + 3f) ratio was observed in the CR-D; this value was statistically greater than all other respiratory muscles. The physiological significance of this finding is unclear. On the other end of the spectrum, the 3 f/(1s + 1f + 3f) ratio was lower in the CO-D compared with that in all other respiratory muscles. Finally, no differences in the 3 f/(1s+ 1f + 3f) ratio existed among the other respiratory muscles.
Because of the high percentage of type 1s MLC, the ratio of MLC 1s/(1s + 1f+ 3f) was significantly higher in the CO-D and CR-D compared with that in all other muscles. No differences existed in the MLC 1s/(1s + 1f + 3f) ratio between other muscles.
Relationships between enzyme activities and MHC phenotype. The correlational relationship between MHC isoforms, MLC isoform ratios, and enzyme activities across all respiratory muscles are shown inTable 4. Several points of interest emerge from these data. First, a significant positive correlation exists between CS activity and%type I MHC,%type IIa MHC, and the ratio of MLC 1s/(1s + 1f + 3f). In contrast, CS activity was inversely related (i.e., significant negative correlation) to LDH activity, mATPase activity, and%type IIb MHC. Finally, a strong positive correlation exists between mATPase activity, and LDH activity, and the%type IIb MHC. Collectively, these correlations confirm that respiratory and locomotor muscles share similar relationships between myosin phenotype and bioenergetic enzyme activities (18).
To our knowledge, this is the first experiment to examine the MHC isoform content, MLC isoform content, as well as the mATPase and bioenergetic enzyme activities of rat inspiratory and expiratory muscles. Since the diaphragm is reported to be the primary ventilatory muscle involved in quiet breathing in many mammals (8), we hypothesized that the CO-D and CR-D would have different metabolic properties (i.e., higher oxidative capacities) compared with those of less active respiratory muscles. Using the activities of CS and the LDH/CS ratio as markers of the metabolic (i.e., oxidative) status of muscle, our data support the notion that the metabolic properties of the CO-D and CR-D differ from other respiratory muscles. Further, the myosin phenotype of the CO-D and CR-D differed significantly from other inspiratory and expiratory muscles as well as the plantaris muscle. Hence, our hypothesis was supported.
Critique of experimental model. Within a given species, the metabolic properties of skeletal muscle can vary according to muscle, developmental status, and state of training(10,11,19-21). We chose to study rats because of their widespread experimental use in investigations of chest wall mechanics as well as ventilatory muscle experiments. By investigating adult rats, we avoided the potential bias associated with metabolic differences in muscle because of age (i.e., development). Further, we consistently selected samples from specific anatomical locations. While we specifically investigated different muscles with known ventilatory involvement, it seems likely that several of these muscles (e.g., RA and SM) also function in nonventilatory activities. Finally, to avoid large inter-animal differences in the level of daily physical activity, animals were housed in standard cages for 3 months before study.
Bioenergetic differences across respiratory muscles. Again, these experiments tested the hypothesis that the CO-D and CR-D would be metabolically different from other respiratory muscles (i.e., PI, II, EI, RA, and SM muscles). This hypothesis evolved from: 1) the knowledge that chronically active skeletal muscles have high oxidative enzyme activities compared with less active muscles (16); and 2) reports that the CO-D and CR-D are chronically active during quiet breathing in mammals whereas other ventilatory muscles have a more limited recruitment(8). Again, our data support the hypothesis since the LDH/CS ratio was lower in the CO-D and CR-D compared with that in all other respiratory muscles (Fig. 3).
Small and insignificant differences existed between LDH/CS ratios of the SM, PS, II, and EI muscles. These findings suggest that these muscles are metabolically similar. The respiratory muscle with the highest LDH/CS activity ratio was the RA. Therefore, compared with the other respiratory muscles in this study, the RA has a low oxidative capacity and a relatively high glycolytic capacity. The relatively low oxidative capacity in the RA implies that by comparison with the other ventilatory muscles studied, the RA is not highly recruited as a ventilatory muscle in sedentary rats.
A question of obvious importance is “what is the physiological significance of differences in oxidative capacities between respiratory muscles”? The answer is that muscles with high oxidative capacities(e.g., costal and crural diaphragm) would exhibit a high capacity for fat utilization and therefore lower rates of muscle glycogen degradation during periods of prolonged activity (10). Also, compared with muscles with low oxidative capacities, respiratory muscles with high oxidative capacities would produce less lactate during periods of high contractile activity (10). Collectively, these changes may delay fatigue during long periods of high intensity contractile activity(10). Hence, it could be predicted that the costal and crural diaphragm would be more fatigue resistant than the other respiratory muscles examined in this investigation.
Variation of myosin isoforms and mATPase activity. The physiological significance of the variation in myofibrillar proteins observed among respiratory muscles is linked to the fact that the myosin isoform profile and mATPase activity of a muscle is closely associated with the maximal shortening velocity(1,4,6,24,27). That is, muscles that contain high fast MHC and MLC content possess high mATPase activities and therefore exhibit high maximal shortening velocities. Although numerous factors contribute to muscle shortening velocity (e.g., muscle length and geometry), there is growing evidence that the function of the multiplicity of skeletal muscle myosin isoforms is to provide a wide range of maximal shortening velocities and also a range of force-velocity relationships(13,14). Therefore, the differences in myosin profiles observed across the respiratory muscles gives rise to chest wall motor units differing in shortening properties which can be selectively recruited to meet the demands of a particular ventilatory or expulsive task. For example, with the respiratory muscles studied in these experiments, the RA contained the highest relative content of fast (type IIb MHC) myosin isoforms. Hence, it seems likely that the RA should possess a high maximal shortening velocity; this would be useful during powerful expulsive behaviors (e.g., coughing).
SUMMARY AND CONCLUSIONS
These experiments characterized the myofibrillar proteins and the bioenergetic profiles of rat inspiratory and expiratory muscles. Of the muscles investigated, the CO-D and CR-D contained the highest oxidative capacity and lowest glycolytic capacity, which supports the notion that the diaphragm is metabolically different from other respiratory muscles. Among the other respiratory muscles investigated, the RA had the lowest oxidative capacity (i.e., CS activity) and the highest content of type IIb MHC. Collectively, our data indicate that wide variations exist in both bioenergetic enzyme activities and myosin isoforms across respiratory muscles. These differences in both bioenergetic enzyme activities and contractile proteins provide the muscles of the respiratory pump with great versatility in terms of contractile properties and metabolic capacities.
This study was supported by a research grant from the American Lung Association-Florida and the American Heart Association-Florida. The authors thank Michael Hughes for his expert technical help.
Address for correspondence: Scott K. Powers, Ph.D., Ed.D., Center for Exercise Science, Room 33, FLG, University of Florida, Gainesville, FL 32611. E-mail: email@example.com.
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Keywords:©1997The American College of Sports Medicine
DIAPHRAGM; LOCOMOTOR MUSCLES; OXIDATIVE CAPACITY; VENTILATION; GLYCOLYTIC CAPACITY