INTRODUCTION

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EARLY POSTNATAL DEVELOPMENT of the diaphragm muscle (Dia_m) is characterized by dramatic transitions in myosin heavy chain (MHC) isoform expression. In the rat Dia_m, during the first 3 postnatal wk, expression of the MHC_neo isoform gradually disappears, whereas expression of MHC_2X and MHC_2B isoforms appears only by day (D)-14 and increases thereafter (9, 10, 12, 13, 31). In previous studies (9, 24, 31, 36) in the rat Dia_m, we reported that the transition from MHC_neo to MHC_2X and MHC_2B isoform expression was associated with changes in muscle contractile properties. With the postnatal increase in MHC_2X and MHC_2B isoform composition, the maximum unloaded shortening velocity [V_o; measured by using the "slack" method (5)] of the Dia_m became faster, and there was an increase in maximum isometric tetanic force [P_o; force per cross-sectional area (CSA)] (9, 24, 31, 36). Given these postnatal changes in V_o and specific force, we hypothesized that the force-velocity relationship and power output of the Dia_m would also change during early postnatal development and that these changes would be associated with the postnatal transition in MHC isoform composition.

During early postnatal development, the Dia_m becomes increasingly more susceptible to fatigue induced by repetitive isometric activation (24, 26, 35, 36). The increase in susceptibility to fatigue of the Dia_m is also associated with an increase in the relative expression of MHC_2X and MHC_2B isoforms (36). Fibers expressing the MHC_2X and MHC_2B isoforms have higher ATP consumption rates (1, 29, 30, 32, 33) and lower oxidative capacities (27, 32, 36), compared with fibers expressing the MHC_slow and MHC_2A isoforms. Thus we hypothesized that the postnatal increase in susceptibility to fatigue of the Dia_m was due, at least in part, to a greater imbalance between ATP consumption and aerobic capacity of fibers expressing the MHC_2X and MHC_2B isoforms (24, 36).

During shortening contractions, ATP consumption rate increases compared with that during isometric activation (6, 11). Thus the imbalance between energy supply and demand should be exaggerated during shortening contractions, and fatigue should be more rapid. Accordingly, Seow and Stephens (20) reported that in the mouse Dia_m fatigue induced by repetitive shortening contractions was more pronounced than that induced by repetitive isometric activation. We hypothesized that, with the postnatal transition to MHC_2X and MHC_2B isoform expression in the Dia_m, fatigue induced by repetitive shortening contractions would become progressively more pronounced. Accordingly, at different postnatal ages, we compared Dia_m fatigue induced by repetitive isometric and isotonic contractions.

	METHODS

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Animal model and in vitro Dia_m preparation. Experiments were performed on male Sprague-Dawley rats at postnatal days 0, 7, 14, 21, and 28 (D-0 to D-28, respectively) and on 84-day-old rats (adults) (n = 8 rats for each age group). Pregnant mothers were received at 14 days gestation, and after parturition the litter size was culled to eight pups. Pups from smaller litters were not studied to assure normal body growth. Body weights of the pups were measured daily. At D-21, the pups were weaned and, thereafter, they were housed two per cage. All experimental procedures were approved by the Institutional Animal Care and Use Committee at Mayo Clinic and were in strict accordance with the American Physiological Society animal care guidelines.

Electrophoretic determination of MHC isoform composition of the Dia_m. Muscle segments were stretched to 1.5 times resting excised muscle length [approximate optimal length (L_o) for muscle force generation in both adults and neonates (15)] and rapidly frozen in isopentane cooled to its melting point by liquid nitrogen. From one-half of the frozen muscle segment, myosin was extracted by scissor mincing. The myosin extracts were centrifuged and supernatants recovered. After overnight storage to allow precipitation of myosin filaments, the solution was again centrifuged, and the pellet was dissolved in sample buffer, boiled, and then stored frozen. MHC isoforms were separated by SDS-PAGE. Specific MHC bands were identified by immunoblotting, as previously described (13, 27). In silver-stained gels, the relative composition of the different MHC isoforms was determined by densitometry (27).

Immunohistochemical determination of MHC isoform expression in single Dia_m fibers. From the other one-half of the frozen muscle segment, five serial transverse sections were cut at 10-µm thickness and reacted with mouse primary antibodies against different MHC isoforms. In some cases, only a single antibody was used, e.g., anti-MHC_all-2X [Schiaffino et al., BF-35 (19), IgG]. However, in most cases, pairs of mouse IgG or IgM primary antibodies were used, e.g., anti-MHC_slow (Novocastra, IgG), anti-MHC_2A [Blau A4.74 (8), IgG or Blau N1.551 (8), IgM], anti-MHC_2B [Schiaffino et al., BF-F3 (19), IgM] and anti-MHC_neo (Novocastra, IgG). Primary antibodies were diluted in PBS containing 0.5% bovine serum albumin (5 mg/ml) and applied to the muscle section for ~2 h at room temperature. Slides were then washed in PBS and reacted with Cy3- or Cy5-conjugated secondary antibodies (goat anti-mouse IgG or goat anti-mouse IgM) for 45 min at room temperature. The use of antibody pairs allowed for double labeling of MHC isoform expression in the same section with minimal cross-reactivity. This was confirmed by adding the opposite secondary antibody to a section incubated with only IgG or IgM primary antibody. Sections incubated with only the secondary antibodies served as controls for nonspecific reactivity of all primary antibodies. With the use of these methods, coexpression of different MHC isoforms could be determined within single fibers, with the exception of MHC_2X isoform coexpression.

CSA of Dia_m fibers. The transverse sections of Dia_m fibers were imaged by using a Bio-Rad (MRC500/600) confocal system mounted on an Olympus BH2 microscope. The imaging system was calibrated for morphometry by using a stage micrometer. With the use of a ×20 microscope objective, each picture element (pixel) in the digitized image had a CSA of 0.15 µm². Approximately 100 fibers were sampled from each Dia_m. The relative contribution of each MHC phenotype to total Dia_m mass was estimated based on the proportion of fibers displaying a given pattern of MHC isoform expression and their average CSA.

Dia_m contractile properties. Two muscle segments from each Dia_m were mounted vertically in glass tissue chambers that were constantly perfused with Rees-Simpson solution [(in mM) 135 Na⁺, 5 K⁺, 2 Ca²⁺, 1 Mg²⁺, 120 Cl, 25 , and 0.012 d-tubocurarine] aerated with 95% O₂-5% CO₂ and maintained at 26°C (pH 7.4). In one muscle segment, isometric fatigue was assessed, and in the other muscle segment, isotonic fatigue was assessed (see below). In the second muscle segment, force-velocity relationships were also characterized (see below). In both muscle segments, the costal margin insertion of the fibers was fixed by using a surgical clamp mounted in series with a micropositioner near the base of the tissue chamber. A small piece of aluminum foil was glued to the central tendon and then attached to a dual-mode length-force servo controller (Cambridge Technologies, model 300B) via a fine aluminum wire. The wire provided a noncompliant attachment of the muscle segment to the force transducer.

2

Isometric and isotonic fatigue properties. Isometric and isotonic fatigue were evaluated simultaneously in two separate muscle segments obtained from each Dia_m. In one muscle segment, isometric fatigue resistance was assessed during repetitive 40-Hz stimulation in 330-ms-duration trains repeated each second for a 5-min period. An isometric fatigue index was calculated as the ratio of force generated after 2 min of stimulation to the initial force.

Statistical analysis. A one-way analysis of variance (age as the grouping variable) was used to evaluate maturational changes in P_o, V_max, maximum power output, maximum work performance, and isometric and isotonic endurance times. A two-way analysis of variance for repeated measures (age and time as grouping variables) was used to evaluate isometric and isotonic fatigue. When appropriate, post hoc analysis (unpaired Student's t-test with Bonferroni correction) was also performed. In all cases, statistical significance was established at the 0.05 level. All data are represented as means ± SE.

	RESULTS

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Postnatal changes in body weight. During early postnatal development, body weights of the male rats progressively increased from 7.4 ± 0.3 g at D-0 to 307.4 ± 3.6 g in adults (P < 0.05). During the first 3 postnatal wk, body weights increased at a rate of ~10 g/wk, whereas the growth rate increased to ~30 g/wk from D-21 to D-84 (adults).

MHC isoform composition of the Dia_m. Based on densitometric analysis of the SDS-PAGE, the relative MHC isoform composition of the Dia_m was determined at different postnatal ages (Table 1). At D-0 and D-7, only MHC_neo, MHC_slow, and MHC_2A were expressed in the Dia_m, with the MHC_neo predominating at both ages. The MHC_2X isoform appeared by D-14, and the MHC_2B isoform by D-21. The MHC_neo isoform was no longer expressed in the Dia_m by D-28. The relative expression of the MHC_slow isoform increased after D-0 and reached adult values by D-14. The relative expression of the MHC_2A isoform also increased after D-0, reaching highest levels between D-14 to D-28, before declining slightly in the adult. After D-14, the relative expression of the MHC_2X isoform increased, reaching adult levels by D-28. The relative expression of the MHC_2B isoform also increased after appearing at D-21.

Immunohistochemical determination of MHC isoform expression in Dia_m fibers. Immunohistochemical analysis was used to provide qualitative information regarding the distribution of MHC isoform expression within single Dia_m fibers at different postnatal ages (Table 2). However, with coexpression of MHC isoforms, immunohistochemistry could not determine the relative amounts of each MHC isoform expressed. In addition, coexpression of the MHC_2X isoform could not be unambiguously determined based on immunohistochemistry.

View larger version (88K): [in this window] [in a new window]   Fig. 1.   Expression of different myosin heavy chain (MHC) isoforms in diaphragm muscle (Diam) fibers was identified by immunoreactivity to specific MHC antibodies. In younger animals, the MHCneo isoform was coexpressed with MHCslow and/or MHC2A isoforms. D, postnatal days; N, neo.

CSA of Dia_m fibers. The increase in body weight during early postnatal development was accompanied by a dramatic increase in the CSA of Dia_m fibers (Table 2). Fiber CSAs in the D-0 Dia_m were relatively uniform, although those fibers coexpressing the MHC_neo and MHC_2A isoforms were smaller than other fibers (P < 0.05; Table 2). In the D-0 Dia_m, fibers expressing the MHC_neo isoform, either alone or in combination with other MHC isoforms, contributed ~85% to total Dia_m mass (Table 2).

Contractile properties. The L_o of Dia_m fibers increased threefold from D-0 to adulthood (P < 0.05; Table 3). Both the P_t and P_o of the Dia_m increased significantly with postnatal maturation (P < 0.05; Table 3). The increase in P_t and P_o was proportionate, such that the P_t / P_o remained relatively constant across postnatal maturation.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Force-velocity relationship of Diam during postnatal development. Maximum shortening velocity [Vmax; in muscle lengths (ML)/s] was estimated by extrapolating force-velocity curves to zero load. With maturation, Vmax became progressively faster.

View larger version (34K): [in this window] [in a new window]   Fig. 3.   Force-power relationship of Diam during postnatal development. With maturation, maximum power of Diam progressively increased.

Isometric and isotonic fatigue properties. During early postnatal development, susceptibility of the Dia_m to fatigue induced by repetitive isometric activation at 40 Hz increased significantly (P < 0.05; Figs. 4, A and B and 5A). At D-0, the Dia_m continued to generate ~80% of initial force after 2 min of repetitive stimulation, whereas in adults the Dia_m generated only ~30% of initial force after 2 min (Figs. 5A and 6A).

View larger version (42K): [in this window] [in a new window]   Fig. 4.   Examples of fatigue induced by repetitive isometric and isotonic contractions of Diam at D-0 (A and C) and in adult (B and D). For isotonic contractions, muscle shortened against a load corresponding to the maximum power for that age. Note the fatigue resistance of D-0 Diam during both isometric and isotonic activation, compared with greater fatigability of the adult Diam. At each age, the Diam was more susceptible to isotonic than to isometric fatigue.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Fatigue during repetitive activation was defined as a decrement in isometric force (A), isotonic shortening velocity (B), and isotonic power output (C). Note that, for each index, fatigue of Diam increased with maturation.

View larger version (28K): [in this window] [in a new window]   Fig. 6.   Isometric fatigue index (%residual force after 2-min repetitive activation) of Diam decreased with maturation (A). Similarly, isotonic endurance time (time for power output to decline to zero) of Diam also decreased with maturation (B).

	DISCUSSION

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The present study examined the association between postnatal transitions in MHC isoform expression and changes in isometric and isotonic contractile and fatigue properties of the rat Dia_m. The disappearance of MHC_neo isoform expression and the emergence of MHC_2X and MHC_2B isoform expression were associated with a maturational increase in P_o, V_max, and maximum power output of the Dia_m. However, with this postnatal increase in contractility, the Dia_m became more susceptible to fatigue, especially during shortening contractions.

In the present study, MHC isoform expression was determined by using both SDS-PAGE and immunohistochemistry. Based on densitometric analysis, the relative composition of different MHC isoforms in the Dia_m could be determined at each postnatal age. However, the distribution of MHC isoform expression within single fibers could not be evaluated by whole muscle SDS-PAGE. In a previous study in the adult Dia_m, we used SDS-PAGE to determine MHC isoform expression in single fibers (32). This method was particularly useful in assessing the relative composition of different MHC isoforms in those fibers coexpressing MHC isoforms. However, because of the small size and fragility of fibers in the developing Dia_m, it was not possible to reliably dissect single fibers and utilize SDS-PAGE analysis in the present study. Instead, we utilized immunohistochemistry to identify MHC isoform expression within single fibers. This method is limited by the fact that the extent of MHC isoform coexpression cannot be quantified. Furthermore, since no specific antibody for the MHC_2X isoform exists, it was not possible to detect coexpression of the MHC_2X isoform. Thus, whereas SDS-PAGE analysis revealed that the MHC_2X isoform was present in the Dia_m at D-14, the expression of this isoform could not be detected by immunohistochemistry until D-28, when it was singularly expressed in some fibers.

The increase in Dia_m P_o with maturation observed in the present study is consistent with several previous studies (9, 15, 24, 31, 35). As we previously reported, the progressive increase in Dia_m P_o with development is inversely correlated with the expression of the MHC_neo isoform and positively correlated with the emergence of MHC_2X and MHC_2B isoform expression (9). In the neonatal Dia_m, P_o is only one-half that of the adult. The lower specific force of the neonatal Dia_m may reflect, at least in part, the higher relative contribution of interstitial space to total muscle area in the neonate (15). However, it is clear that other factors must also contribute to the lower P_o of the neonatal Dia_m. For example, it is possible that Dia_m fibers during early postnatal development have a lower myofibrillar volume density than do adult fibers (9, 14, 24, 31, 35). It is also possible that neonatal Dia_m fibers differ in the force per cross bridge or in cross-bridge cycling kinetics.

Several previous studies have demonstrated a relationship between MHC isoform expression and the V_o or V_max of single muscle fibers (2, 4, 16-18, 34). Generally, studies in adult animals have demonstrated that muscle fibers expressing fast MHC isoforms have faster V_o or V_max than fibers expressing the MHC_slow isoform. In the adult rat Dia_m, Eddinger and Moss (4) reported that the V_o of fibers expressing fast MHC isoforms was ~3.5 times faster than that of fibers expressing the MHC_slow isoform. In developing rat soleus and psoas muscle fibers, Reiser and colleagues (16, 17) reported that the V_o of fibers expressing developmental MHC isoforms was slower than that of fibers expressing fast MHC isoforms but faster than that of fibers expressing the MHC_slow isoform.

In previous studies in the rat Dia_m (9, 31), we found that postnatal transitions in MHC isoform composition strongly correlated with an increase in V_o, as determined by using the slack method (5). During the first 3 postnatal wk, the increase in V_o of the Dia_m inversely correlated with the decrease in the relative contribution of the MHC_neo isoform. After D-14, the increase in V_o positively correlated with the progressive increase in MHC_2X and MHC_2B isoform expression. In these previous studies, the slack test was used to determine V_o, whereas in the present study V_max was estimated by extrapolation of the force-velocity relationship to zero load. Moreover, measurements were obtained at 26°C rather than 15°C as in our previous studies (9, 31). In muscles consisting of different fiber types, such as the Dia_m, it has been suggested that V_max more accurately reflects the relative composition of different MHC isoforms while V_o may reflect primarily the fastest fibers (3). Despite the differences in technique, the increase in V_max that we observed during early postnatal development of the Dia_m in the present study was qualitatively similar to that previously observed. However, if a Q₁₀ of ~2 is assumed for V_o measurements, the corrected V_o for the Dia_m at 26°C would be considerably faster than the V_max observed in the present study at each postnatal age. For example, at D-0, the V_max at 26°C observed in the present study was 1.2 ML/s, whereas the temperature-corrected V_o of the Dia_m at D-0 was 2.0 ML/s. In the adult Dia_m, this discrepancy between V_o and V_max would be even more pronounced; a V_max of 5.1 ML/s in the present study vs. a temperature-corrected V_o of 12.6 ML/s. The greater discrepancy between V_o and V_max in the adult most likely reflects the contribution of fibers expressing the MHC_2X and MHC_2B isoforms.

As previously observed, susceptibility of the Dia_m to fatigue induced by repetitive isometric activation increases during early postnatal development (14, 15, 24, 35, 36). This age-related increase in the susceptibility of the Dia_m to fatigue cannot be explained by maturational changes in fiber oxidative capacity, since succinate dehydrogenase activity and capillary density of Dia_m muscle fibers actually increase during the early phase of postnatal development when fatigue resistance is declining (22-24, 36). In the adult Dia_m, the capacity for oxidative phosphorylation in fibers expressing the MHC_slow and MHC_2A isoforms remains relatively high, whereas the oxidative capacity of fibers expressing the MHC_2X and MHC_2B isoforms is substantially lower (32). These fiber type differences in oxidative capacity in the adult do correspond with the fatigue resistance of the motor units that they comprise (27).

Differences in ATP consumption rate can also contribute to the susceptibility of different muscle fiber types to fatigue. The MHC is the site of hydrolysis of ATP during cross-bridge cycling, i.e., actomyosin ATPase. Recent studies have clearly demonstrated an association between MHC isoform expression, actomyosin ATPase activity, and V_max in adult muscle fibers (1, 29, 30, 33). Using a quantitative histochemical technique to measure actomyosin ATPase activity of muscle fibers in the adult rat Dia_m, we also found that fibers expressing the MHC_slow isoform have the lowest actomyosin ATPase activity, followed in rank order by fibers expressing MHC_2A, MHC_2X, and MHC_2B isoforms (32). Furthermore, in a recent study, we found that the actomyosin ATPase activity of fibers coexpressing the MHC_neo isoform with either MHC_slow or MHC_2A was lower than that of fibers expressing adult fast MHC isoforms (28). These fiber type differences in actomyosin ATPase activities support the hypothesis that postnatal changes in energy demands of cross-bridge cycling may account, at least in part, for maturational changes in fatigue resistance (24, 36). The slower cross-bridge cycling rate and lower ATP consumption of fibers expressing the MHC_neo isoform, together with a higher oxidative capacity, may lead to a better balance between energy supply and utilization and thus a lower susceptibility to fatigue. In contrast, the faster cross-bridge cycling rate and higher ATP consumption of fibers expressing the MHC_2X and MHC_2B isoforms, together with their lower oxidative capacity, may lead to an imbalance between the energy supply and utilization and thus a greater susceptibility to fatigue.

The observation that the Dia_m was more susceptible to fatigue during repetitive isotonic shortening contractions compared with repetitive isometric activation is consistent with the previous study of Seow and Stephens (20) in the mouse Dia_m. Because with shortening contractions ATP consumption rate increases (6, 11), these results also support the hypothesis that fatigue results, at least in part, from an imbalance between energy supply and demand. The fact that the difference between isotonic and isometric fatigue becomes increasingly more pronounced during postnatal development is consistent with the higher ATP consumption rates and lower oxidative capacity of fiber expressing the MHC_2X and MHC_2B isoforms, which emerge only after D-14 in the rat Dia_m.

Because of more compliant lung and chest wall mechanics, it has been proposed that recruitment of a greater fraction of Dia_m fibers is required to sustain normal ventilation in the neonate (24). Accordingly, polyneuronal innervation of rat Dia_m fibers until D-14 facilitates the more complete recruitment of fibers during ventilatory behaviors (24). In contrast, in the adult Dia_m, it is likely that recruitment of more fatigable motor units consisting of type IIX and IIB fibers is not required to accomplish normal ventilatory behaviors (21, 25). With maturation and the differentiation of fibers expressing MHC_2X and MHC_2B isoforms, the functional reserve capacity of the diaphragm increases (i.e., a greater difference between maximum force vs. force generation during ventilation) (21). However, because of the increased susceptibility of type IIX and IIB fibers to fatigue, it is unlikely that these fibers are recruited during normal ventilation. Thus the normal postnatal transitions in MHC isoform expression in the Dia_m most likely have little impact on sustaining ventilation. Instead, the transition to MHC_2X and MHC_2B isoform expression allows for a greater diversity of Dia_m motor behaviors, especially those requiring shorter duration activation with greater power output.