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Division of Pulmonary/Critical Care Medicine, Department of Medicine, Cedars-Sinai Medical Center Burns and Allen Research Institute, Cedars-Sinai Medical Center, University of California, Los Angeles School of Medicine, Los Angeles, California 90048; and Departments of Anesthesiology and Physiology and Biophysics, Mayo Clinic, Rochester, Minnesota 55905
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
ABSTRACT 
Lewis, Michael I., Thomas J. Lorusso, Wen-Zhi Zhan, and Gary
C. Sieck. Interactive effects of denervation and malnutrition on
diaphragm structure and function. J. Appl.
Physiol. 81(5): 2165-2172, 1996.
The purpose of
this study was to examine the interactive effects of unilateral
denervation (DN) and prolonged malnutrition (MN) on the structure and
function of the diaphragm muscle (Dia). Four groups of
rats were studied: control (Con), MN, DN, and DN-MN. MN
began 2 wk after DN and lasted 4 wk. In both the DN and DN-MN groups,
the relative loss in Dia weight exceeded the relative change in body
weight. Compared with the Con group, Dia specific force was reduced by
~40% in both the DN and DN-MN groups but was unaffected in the MN
group. Dia fatigue resistance improved in all experimental groups but
to a greater extent in the DN and DN-MN groups. In both the DN and
DN-MN groups, ~50% of Dia fibers were classified as type IIc,
whereas fiber type proportions did not change in the MN group. In the
DN group, only type IIb/x fibers atrophied, whereas all fiber types
atrophied in the MN and DN-MN groups. We conclude that in the DN-MN
group the reduction in specific force combined with the reduction in total cross-sectional area of the muscle significantly curtails Dia
force-generating capacity.
diaphragm contractility; diaphragm fiber type proportions; diaphragm fiber cross-sectional area
INTRODUCTION BOTH PROLONGED INACTIVITY of the diaphragm muscle (Dia)
and malnutrition (MN) adversely affect the structure and function of
the muscle. This may have important clinical implications because both
conditions may coexist, particularly in critically ill patients. Inactivity and MN differ considerably with regard to the physiological and morphometric sequelae induced in the Dia. For example, Dia inactivity induced by unilateral denervation (DN), tetrodotoxin (TTX)
nerve blockade, or spinal isolation results in a significant reduction
in Dia specific force [i.e., force/unit cross-sectional area
(CSA)] (22, 33) that begins as early as 3 days after DN (13) and
then persists (22, 33). In contrast, Dia specific force is preserved
after both acute (19) and prolonged MN (17, 20). In both conditions,
Dia fatigue resistance improves (20, 27, 34). With MN, Dia weight is
reduced proportionately to body weight and there is an atrophy of all
fiber types, although the atrophy of type II fibers is greater than
that of type I fibers (20, 27). After 2 wk of DN or TTX nerve blockade,
type I and IIa fibers in the rat Dia hypertrophy, whereas type IIb/x
fibers atrophy (22). However, it is unknown to what extent the factors promoting hypertrophy of type I and IIa fibers after DN might offset
the negative protein turnover and atrophy of these fibers induced by
MN. Similarly, it is unknown whether and to what extent the atrophy of
type IIx and IIb fibers induced by DN would be compounded by the
additional insult of MN. In addition, the presence of
"transitional" type IIc fibers, which coexpress slow and fast myosin heavy chain (MHC) isoforms, have been reported in the Dia after
DN (11). What impact MN might have on these transitional fibers is presently unknown.
With both MN and DN, the total force-generating capacity of the Dia
would be expected to fall, although the pathophysiological mechanisms
contributing to this decline would differ. After DN, both the decline
in specific force and a reduction in the CSA of type IIb/x fibers would
lead to a decrease in total Dia force-generating capacity, whereas with
MN, only a reduction in the total CSA of the Dia would contribute. We
hypothesize that DN and MN have an additive negative impact on Dia
function because, in addition to a reduction in CSA, Dia specific force
would also be reduced. This combination of effects may result in a
catastrophic loss of force-generating capacity of the whole Dia.
METHODS Animals. Forty adult male
Sprague-Dawley rats were studied (initial body weight 356 ± 3 g).
The animals were divided into four groups:
1) control (Con;
n = 10),
2) DN
(n = 10),
3) MN
(n = 11), and
4) DN-MN
(n = 9). The body weights of the MN
animals were adjusted by alterations in dietary intake to match those of the DN-MN animals (i.e., pair weighting of the MN and DN-MN groups).
All animals were housed individually. The ambient temperature in the
vivarium was maintained at 22°C, and the light cycle was fixed at
12:12-h on-off.
DN. Survival surgery was performed
under general anesthesia induced by an intramuscular injection of
ketamine (100 mg/kg) and xylazine (10 mg/kg). The main trunk of the
right phrenic nerve was dissected in the neck, and an ~3-mm segment
of nerve was transected and removed to avoid possible reinnervation of
the Dia. Immediately after phrenic nerve transection, asymmetrical
movements of the abdominal wall were observed during inspiration. In a
separate group of DN animals, complete paralysis of the right Dia was
confirmed by the absence of electromyographic activity during
inspiration. In the Con and MN groups, sham surgery was performed to
mimic that in the DN group. In the present study, terminal experiments were performed 6 wk after DN.
Nutritional deprivation paradigm. MN
was induced by reducing the daily intake of Purina Rat Chow (56%
carbohydrate, 23% protein, 4.5% fat, 6% fiber, and 10.5% ash and
minerals) to approximately one-third of the estimated daily consumption
of the Con animals. Water was provided ad libitum to all groups. The
nutritional deprivation protocol was begun 2 wk after DN and lasted 4 wk.
In vitro measurement of Dia contractile and fatigue
properties. The techniques utilized in determining the
in vitro isometric contractile and fatigue properties of the Dia have
been previously reported (20, 27). After the induction of deep
anesthesia (pentobarbital sodium 20 mg/kg ip), the Dia was rapidly
excised, and a narrow strip (4-5 mm) was dissected from the right
midcostal region, maintaining the fiber attachments to the ribs and
central tendon intact. The Dia strip was mounted vertically in a tissue bath containing Krebs-Henseleit solution maintained at 26°C and constantly aerated with 95% O2
and 5% CO2. The plastic clamp at the costal margin was attached to a calibrated force transducer (Grass
FT10), and a separate clamp at the costal margin was attached to a
micromanipulator (Kopf).
Muscle segments were stimulated directly with ~1- to 2-ms-duration
monophasic pulses at supramaximal intensity (Grass S88 stimulator).
D-Tubocurare (12 µmol/l) was
added to the tissue bath to block neuromuscular transmission. During
single-pulse stimulations, the muscle length was adjusted until maximum
twitch force responses were obtained. Isometric contractile and fatigue properties were then determined at this optimal length
(Lo), which was
measured with a digital caliper (accurate to 1 µm; Mitutoyo).
Peak twitch force (Pt),
contraction time (time to Pt),
and half-relaxation time (RT1/2;
time for Pt to fall to one-half of maximum) were determined from a series of single pulses.
Force-frequency relationships were measured for stimulus frequencies
ranging from 5 to 100 Hz in trains of 1-s duration. At least 30 s
intervened between each stimulus train.
Pt and maximum tetanic forces
(Po) were normalized for
estimated CSA of the muscle [CSA = muscle weight (in g)/1.056
g/cm3 × Lo (in
cm)]. Wet weight of the right hemidiaphragm was determined after
removal of any Krebs solution by blotting and removal of attached
central tendon and ribs.
Fatigue resistance of the Dia was determined with a fatigue test in
which repetitive stimuli were presented over a 2-min period (40 Hz in
trains of 330 ms repeated each second). A fatigue index (FI) was
calculated as the ratio of the force generated at 2 min to the initial
tetanic force.
Histochemical procedures: fiber type proportions and
CSA. After physiological measurements were made, the
muscle segment and an adjacent segment were stretched to
Lo, as determined
from physiological measurements, mounted on cork, and rapidly frozen in
isopentane cooled to its melting point by liquid nitrogen. Serial cross
sections of the Dia fibers were cut at 6-µm thickness with a cryostat
(Reichert-Jung) kept at Dia muscle fiber CSA was determined from microscopic (Olympus BH-2)
images of the muscle sections digitized into an array of 1,024 × 1,024 pixels (picture elements) with a computer-based image-processing
system (MegaVision 1024 XM). A microscope stage micrometer was used to
calibrate the image-processing system for morphometry. By using a
×20 microscope objective, the area of each pixel was 0.15 µm2. The CSA of individual
fibers was determined from the number of pixels within outlined fiber
boundaries.
Statistical analysis. Statistical
analysis was performed with an analysis of variance (ANOVA). With a
two-way ANOVA, the two experimental factors were innervation status
(levels: presence or absence of DN) and nutritional status (levels:
presence or absence of MN). Post hoc analysis (Newman-Keuls test) was
used after a one-way ANOVA to compare differences between pairs of independent groups. These specific contrasts were determined, particularly if a significant interaction was observed, with the two-way ANOVA. In comparing force-frequency relationships (the eight
frequency points) and progressive force loss during the fatigue test
(the four 30-s time periods), ANOVA with repeated measures was employed
within the framework of the two-factor design above. Paired
t-tests were used to compare initial
and final body weights within groups. An RESULTS Body and Dia weights. Over the 6-wk
experimental period, Con animals progressively increased body weight
(+37.4%; Fig.
1A), whereas the body weight gain of free-eating DN animals was
significantly attenuated (+21.6%; P < 0.01). After the MN paradigm, the weights of DN-MN animals
decreased significantly (
The ratio of hemidiaphragm weight to body weight is presented in Table
1. In MN animals, there was a proportionate
reduction in body and Dia weights, and thus the ratios of the Con and
MN animals were similar. In DN animals, however, hemidiaphragm weight was reduced disproportionately to body weight, resulting in a significant reduction in the ratio compared with the MN and Con animals
(Table 1; P < 0.01). In DN-MN
animals, hemidiaphragm weight was also disproportionately reduced
compared with body weight but not to the extent noted with DN alone
(Table 1; P < 0.05).
Table 1.
Ratio of hemidiaphragm to body weight
20°C and stained for myofibrillar
adenosinetriphosphatase (mATPase) after preincubation at various pH
levels (2). Dia fibers were classified as type I or II on the basis of
differences in staining intensity for mATPase after alkaline (pH = 9.0)
preincubation; type I fibers stain lightly for mATPase, whereas type II
fibers stain darkly. After preincubation at pH 4.3, these staining
patterns for mATPase were reversed except for fibers classified as type IIc. Type II fibers were further subclassified as types IIa or IIb/x on
the basis of staining profiles for mATPase after preincubation at pH
4.55. The histochemical classification of these fiber types in the Dia
corresponds to the expression of different MHC isoforms (28). However,
the histochemical procedure that was used could not distinguish between
fibers expressing MHC-2B and -2X isoforms (28). Indeed, in the rat Dia,
we found that most fibers classified as type IIb with standard
histochemical techniques actually express the MHC-2X isoform either
alone or in combination with the MHC-2B isoform (28). Therefore,
because we did not distinguish between type IIb and IIx fibers in the
present study, we have used the term IIb/x to more accurately classify
these fibers. Fiber type proportions were determined from a sample of
~200-300 fibers from each muscle.
level of 0.05 was used to
determine significance. All data are reported as means ± SE.
33.2%;
P < 0.01), which was tracked by the
MN animals (33.1%). As depicted in Fig.
1B, right hemidiaphragm weight was
significantly reduced by DN alone and in the MN group compared with
that in the Con group (36.8 and 50.4%, respectively;
P < 0.001). After MN, right
hemidiaphragm weight was further reduced in the DN-MN animals
(61.5%; P < 0.001). The
decrement in hemidiaphragm weight (referenced to the Con hemidiaphragm) was greater in DN-MN animals compared with the MN animals (61.5 vs. 50.4%; P < 0.01). Thus a
significant effect was noted with both DN and MN alone
(P < 0.001), and a significant
interaction was noted between DN and MN
(P < 0.001).
Fig. 1.
A: initial (open bars) and final body
weights (solid bars) in control (Con), malnourished (MN), denervated
(DN), and DN-MN groups. Note: in DN group, body weight gain was
attenuated. A marked reduction in body weight was observed in DN-MN
(and MN) group. B: diaphragm weights
in same groups. Note: although a significant reduction in weight of
right hemidiaphragm was noted in DN group, a further significant
reduction was observed in DN-MN group. Values are means ± SE.
* Significant difference from Con group.
+ Significant difference
from DN group. # Significant
difference from MN group.
[View Larger Version of this Image (85K GIF file)]
Group
Ratio
Con
0.862 ± 0.031
MN
0.876 ± 0.023
DN
0.664 ± 0.027*
DN-MN
0.766 ± 0.025*
Values are means ± SE. Con, control; MN, pairweight
malnourished; DN, denervated; DN-MN, denervated + malnourished. Ratio, g diaphragm weight/kg body weight.
*
Significantly different from
Con.
Significantly different from DN.
Isometric contractile and fatigue
properties. The
Lo values of Dia
segments from the midcostal region were similar across groups (Table
2). Compared with Con animals,
Pt was significantly reduced in DN
(41%) and DN-MN animals (33%;
P < 0.001) but not in MN animal.
Twitch contraction time was prolonged only in DN-MN animals
(P < 0.001; Table 2). In contrast,
twitch RT1/2 was prolonged in both
the MN and DN animals compared with the Con animal
(P < 0.01; Table 2). In the DN-MN
animals, twitch RT1/2 was even further prolonged compared with DN and MN animals
(P < 0.01; Table 2).
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The force-frequency relationship in the MN animals was comparable to
that of the Con animals (Fig.
2A). In
contrast, there was a significant reduction in force (expressed in
N/cm2) at all stimulation
frequencies for both the DN and DN-MN groups compared with the Con and
MN groups (P < 0.001; Fig
2A). As a result,
Po was significantly reduced (in
DN and DN-MN animals (39-43%; P < 0.001; Fig. 2A). There was no
difference between the DN and DN-MN groups with respect to the
reduction in Po (39 and
43%, respectively). Thus the reduced specific force was related to the effect of DN, with no interaction between DN and MN. Although there was a trend for the force-frequency relationships to be shifted
to the left at low frequencies in all groups, this leftward shift was
significant for only the DN-MN groups
(P < 0.05; Fig. 2B).
Improved fatigue resistance of the Dia was noted for all experimental
groups compared with the Con group, denoting significant effects for
both the DN and MN groups (Fig. 3; Table 2;
P < 0.01). In the DN and DN-MN
groups, the fatigue resistance of the Dia was higher than that observed
in the MN group (P < 0.01; Fig. 3).
There were no differences in fatigue resistance between the DN and
DN-MN groups (Fig. 3). The residual forces at the end of the 2-min
fatigue tests need to be considered in view of the differences in
specific forces of the Dia in the various groups (i.e., preserved in
Con and MN groups, whereas significantly reduced in DN and DN-MN
groups). In Con animals, the residual force was 6.5 ± 0.4 N/cm2. In the MN group (where
specific force was not reduced, but fatigue resistance improved), the
residual force was significantly greater than in the Con group (8.8 ± 0.5 N/cm2;
P < 0.01). In the DN and
DN-MN animals, despite a reduced specific force, the residual forces
after 2 min of repetitive stimulation were maintained (7.5 ± 0.7 and 7.4 ± 0.8 N/cm2,
respectively). Because the
Lo of muscle
fibers was not changed in any of the experimental groups, mass is a
reflection of muscle CSA available for force generation. Thus,
considering that muscle mass was reduced in each of the three
experimental models (i.e., the total CSA available for force generation
was less), it was estimated that the muscle force-generating capacity
of the Dia after the fatigue test was reduced by ~34% in the MN
animals, ~27% in the DN animals, and ~57% in the DN-MN animals
compared with the Con animals.
Fiber type proportions. Fiber type proportions in the MN group were comparable to those found in the Con group (Table 3). In the DN and DN-MN animals, the proportions of type I, IIa, and IIb/x fibers were all significantly reduced compared with the Con and MN animals, indicating a significant effect of DN only, with no interaction between DN and MN (P < 0.01; Table 3). This reduction in fiber type proportion was the result of a marked increase in the proportion of type IIc fibers, which accounted for ~50% of all fibers in both the DN and DN-MN groups (Table 3).
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Fiber CSA. In the MN group, there was
significant atrophy of all fiber types
(P < 0.01; Fig.
4). The greatest impact was evident in type
IIb/x fibers (~64%), with less atrophy of type I (47%; P < 0.01) and type IIa fibers
(53%). In the DN animals, only type IIb/x fibers atrophied
compared with the Con animals (P < 0.01), whereas the CSAs of type I and IIa fibers were similar to those
of Con animals (Fig. 4). The atrophy of type IIb/x fibers was
comparable to that observed in the MN group (i.e., 58% and 64%, respectively). In the DN-MN group, type I and IIa fibers atrophied compared with Con and DN animals
(P < 0.01; Fig. 4). The further
atrophy of type IIb/x fibers was also significantly greater than that
noted in the DN group alone, indicating a significant interaction
between DN and MN for those fibers (P < 0.05; Fig. 4). The CSA of type IIc fibers was also significantly
smaller in the DN-MN group compared with the DN group (44%;
P < 0.01; Fig. 4).
The relative contributions of type I, IIa, IIb/x, and IIc fibers to the total CSA of the Dia was calculated based on fiber type proportions and mean CSA of each fiber type. In the MN group, there were no significant changes in the relative contributions of the different fiber types to total Dia area (Table 4). In the DN and DN-MN groups, the appearance of type IIc fibers caused a significant reduction in the relative contribution of type IIb/x fibers (P < 0.01) despite a significant reduction in the proportion of type I and IIa fibers (Table 4). In DN and DN-MN animals, the relative contribution of type IIc fibers to total muscle area was ~50% (Table 4). No differences in the relative contribution of the different fiber types to total Dia area (Table 4) were observed between the DN and DN-MN animals.
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DISCUSSION
The present study demonstrated that the combined effects of prolonged DN and MN are additive and are likely to have a serious impact on the functional reserve capacity of the Dia. The reduction in Dia specific force after DN was neither improved nor aggravated with the imposition of MN. However, in the DN-MN group, there was significant atrophy of all fiber types compared with atrophy of only type IIb/x fibers with DN alone. Indeed, the atrophy of type IIb/x fibers was exacerbated in the DN-MN group compared with the DN alone group. In addition, DN-MN animals exhibited a further reduction in the CSA of transitional type IIc fibers compared with the DN alone group. Thus, in the DN-MN Dia, the total CSA available for force generation was reduced compared with the DN alone Dia. The considerable impact of combining DN and MN on fiber morphometry together with a persistent reduction in specific force would be expected to have serious functional sequelae in the DN-MN group.
Critique of methods. In the present study, the experimental paradigm used was geared toward minimizing morbidity and/or mortality with the introduction of a severe nutritional insult in DN animals. Thus MN was imposed only 2 wk after the induction of DN and lasted 4 wk, which allowed a gradual reduction in body weight with no mortality. From our experience, too vigorous a MN regimen or beginning both challenges together would probably have been too stressful for the animals, with an associated high mortality. Although 6 wk of DN might be interpreted as outside a clinically relevant range, we propose that the timing and/or duration of DN is not crucial because similar reductions in Dia specific force can occur as early as 3 days after DN (13). Thus the direction of change with regard to isometric contractile properties is similar at various time intervals after DN (13, 22, 33), which allows one to assess the impact of a superimposed nutritional insult at different time intervals with strong inference.
In the present study, we employed unilateral rather than bilateral DN of the Dia. It has been suggested that the continued inspiratory-related contractions of the contralateral hemidiaphragm may impose passive stretch of the paralyzed side and thereby induce structural and functional adaptations apart from inactivity and DN itself. To explore this possiblility, we implanted sonomicrometry crystals into the midcostal and sternal regions of the unilaterally DN right hemidiaphragm of rabbits (33). We found that the midcostal region of the DN hemidiaphragm displayed a small amount of passive stretch, whereas the sternal region was passively shortened during inspiratory activation of the contralateral hemidiaphragm. In both regions, these passive length changes of the DN hemidiaphragm did not impose any significant mechanical stress on muscle fibers. After 4 wk of DN, we found no significant differences in the regional adaptations of the rabbit Dia, which strongly suggest that passive length changes and mechanical stress are not the main determinants of the contractile and morphometric adaptations induced by unilateral DN.
Fiber type proportions. MN had no significant impact on the proportion of Dia fibers. However, in both DN and DN-MN animals, there was an abundance of type IIc fibers in the Dia. Gauthier and Hobbs (11) demonstrated an increased proportion of type IIc fibers in the rat Dia after 4 wk of DN. In addition, these authors observed that after 8 wk of DN, there was coexpression of both slow and fast MHC isoforms within individual fibers that correlated with histochemical classification of type IIc fibers (11). Similarly, Carraro et al. (4) also demonstrated coexpression of fast and slow MHC isoforms in single fibers from the rat Dia 3-16 mo after DN. Recently, Sieck et al. (28) also observed coexpression of slow and fast MHC isoforms in rat Dia fibers classified histochemically as type IIc. Schiaffiano et al. (24) reported reexpression of embryonic and neonatal MHC isoforms in type 2A fibers of rat hind limb muscles 3-7 days after DN. Gosselin et al. (13) recently demonstrated increased satellite cell mitotic activity in the rat Dia after DN. It is possible that satellite cell activation may be an important factor initiating transitions in MHC isoform expression, resulting in coexpression of isoforms within single fibers.
Fiber CSA. Dia remodeling after DN appears to be a dynamic process. Transient hypertrophy of all muscle fibers occurs during the first week after DN (13, 21, 31). This hypertrophic response is likely the result of increased myofibrillar protein synthesis and content (12) associated with increased RNA and DNA synthesis (21, 32). After the initial hypertrophic phase, atrophy of fibers has been noted, affecting predominantly type II fibers (10). The results of the present study confirm the selective atrophy of type IIb/x fibers after 6 wk of DN. The CSAs of type I and IIa fibers were similar to those of Con fibers. Previously, Miyata et al. (22) found that type I and IIa fibers in the rat Dia hypertrophy immediately after DN and that the CSA of these fibers remains greater than that of Con fibers after 2 wk. In the hamster and rabbit Dia, type I fibers remain hypertrophied 4 wk after DN (33). Yellin (31) reported that after DN white fibers (presumably type IIb/x fibers) in the rat Dia underwent atrophy earlier than in red fibers (presumptive type I and IIa fibers). However, he noted atrophy of red fibers by 2 wk after DN.
The mechanisms underlying the selective morphological adaptations of different fiber types in the rat Dia after DN are unclear. It has been postulated that the initial hypertrophic response may result from the effect of passive stretch imposed on the paralyzed hemidiaphragm by the continued inspiratory-related contractions of the intact contralateral side (12, 21, 31, 32). However, as mentioned previously, Miyata et al. (22) found that in the rabbit Dia continued inspiratory-related activation of the contralateral side after unilateral DN did not impose any significant mechanical stress on the midcostal region. Zhan et al. (33) also found that type I fibers hypertrophied to a similar extent in both the sternal and midcostal regions despite passive lengthening of fibers in the midcostal region and passive shortening of fibers in the sternal region. It is thus likely that removal of neurotrophic influences and/or inactivity differentially affects fiber types in the rat Dia.
In the present study, significant atrophy of all fiber types was noted in DN-MN animals. Of note, a significant reduction in the CSA of type IIc fibers was evident compared with DN fibers, as well as further atrophy of type IIb/x fibers. DN is associated with both insulin resistance of the DN muscle itself, which may reduce amino acid uptake and utilization (3, 29), and increased sensitivity to circulating glucocorticoids due to an upregulation of cytosolic glucocorticoid receptors (6). MN and DN may interact in that protein turnover is further shifted toward net catabolism due to reduced protein synthesis and enhanced proteolysis (25). Severe prolonged MN may also produce enhanced effects in DN muscle because of low circulating levels of insulin (8), insulin-like growth factor I (15), and enhanced levels of circulating glucocorticoids (5). In this regard, Almon and DuBois (1) reported that acute MN increased the degree of atrophy in a variety of DN limb muscles in the rat, whereas the degree of atrophy in these DN muscles was not enhanced in adrenalectomized animals subjected to a similar nutritional deprivation protocol. In addition, both DN itself or a lack of insulin or amino acid availability as might occur with undernutrition has been associated with increased activity of lysosomal proteases and other proteolytic systems within muscle cells that could enhance contractile protein breakdown (9).
In the present study, MN was only initiated after 2 wk of DN, at a time when the initial period of increased protein synthesis had subsided. It is likely that, if MN began just after DN, the interactive effects might have been even more severe because the early hypertrophic responses in the DN Dia might have been blocked by MN.
Contractile and fatigue properties. The observation that twitch RT1/2 was prolonged in both the MN and DN animals is consistent with previous reports (8, 34). Twitch RT1/2 was further prolonged in DN-MN animals. The mechanism(s) underlying the prolongation of RT1/2 in MN animals may relate, in part, to an increase in the relative contribution of type I fibers to total Dia area. However, such an explanation cannot account for prolongation of twitch RT1/2 in the DN and DN-MN groups. In these animals, the prolongation of twitch RT1/2 most likely relates, in part, to the increased contribution of type IIc fibers that have been reported to have slower twitch kinetics.
In each experimental group, alterations in Ca2+ release and sequestration may play a role. Recently, reduced concentrations of parvalbumin, a Ca2+ binding protein associated with muscle relaxation, have been reported in the extensor digitorum longus muscle of the rat after DN (23). Because inverse correlations between parvalbumin and RT1/2 have been reported (14), a reduced parvalbumin level may also partially explain the prolonged RT1/2 observed in the present study. In addition, Schulte et al. (26) reported reduced mRNA and protein expression of specific isoforms of an important sarcotubular protein, sarco(endo)plasmic reticulum Ca2+-ATPase, in soleus and extensor digitorum longus muscles after DN. Associated with a decrease in Ca2+-pump density, these investigators reported a slowing of relaxation and a leftward shift in the force-frequency curve in DN muscles.
The reduction in specific force noted in the Dia after DN was not exacerbated or improved by MN. To the extent that the relative contribution of fiber types to total Dia area was similar between DN and DN-MN animals, one would not have expected an alteration in specific force in the DN-MN Dia. The factors accounting for the reduced specific force with DN are not entirely known and may vary temporarily. For example, reduced Dia specific force has been noted as early as 3 days after DN, during the hypertrophic phase (13). In this instance, muscle fiber injury, demonstrated by ultrastructural analysis, may be an important pathogenic mechanism. The extent to which muscle injury and/or other factors such as reduced myofibrillar density (6), alterations in MHC and myosin light chain expression, disturbances in Ca2+ kinetics, or sodium conductance (16) play a role are unknown and were not evaluated in the present study. It is unlikely that reexpression of embryonic and neonatal MHC isoforms played a role because they are reexpressed in very small amounts (<1%) (24). The reduced specific force, however, when coupled with the significant loss of contractile tissue mass in DN-MN animals, would suggest that total force output of the Dia would be markedly reduced.
Our group previously reported improved fatigue resistance in the hamster Dia after 2 wk of DN (34) or after prolonged MN in the rat (20, 27). In the present study, enhanced fatigue resistance was confirmed in these instances, although the FI was greater in DN than in MN animals. In the DN-MN animals, the FI was not further affected. Although the residual specific force at the end of the fatigue test in the DN and DN-MN animals was not statistically different from that of the Con animals, the total force production of the Dia would be expected to be significantly less, particularly in DN-MN animals, because of a marked reduction in muscle mass (estimated to be ~43% that of Con animals). This would greatly impact on the reserve capacity of the Dia in DN-MN animals. The mechanisms underlying the enhanced fatigue resistance of the Dia in the different groups are unclear, but it is unlikely that these mechanisms are similar across experimental models. In previous studies of prolonged MN, the improved fatigue resistance was explained, in part, by a reduction in the contribution of low-oxidative fibers to total Dia area (27). The neonatal Dia, which contains an abundance of type IIc fibers, is more fatigue resistant than the normal adult muscle (30). Therefore, the improved fatigue resistance of DN and DN-MN animals may relate to the marked increase in the relative contribution of type IIc fibers and the concomitant decrease in type IIb/x fibers.
Clinical implications. The DN model used this study may provide some insights into the qualitative changes and direction of change in Dia structure and function common to many models of functional inactivation. For example, in a previous study, Zhan and Sieck (34) found that the effects of DN on the Dia and medial gastrocnemius muscle of the hamster were qualitatively similar to those induced by TTX nerve blockade. Yet, it is important to note that systemic neuromuscular blockade and/or controlled mechanical ventilation differ significantly from the present model. With DN, communication between phrenic motoneuron and Dia fibers is disrupted, whereas neurotrophic influences are maintained in patients placed on mechanical ventilation. However, in this regard, it is of interest to note that reduced specific force of the Dia was reported in rats after 48 h of controlled mechanical ventilation (18). A similar reduction in specific force of the Dia occurs after short-term (i.e., 3-day) unilateral DN (13).
In summary, DN and MN negatively interact in that Dia specific force is reduced (effect of DN) and type I and IIa fibers atrophy (MN effect) in the DN-MN animals. Together, these effects in the DN-MN animals would markedly reduce the total force-generating capacity of the Dia and thereby decrease the functional reserve capacity of the muscle. Such an effect in the DN-MN animals may be catastrophic in conditions of increased ventilatory demand.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge the superb assistance of Ling Tang, Y. H. Fang, Darlene Ford, and Dr. Janet Elashoff.
FOOTNOTES
Address for reprint requests: M. I. Lewis, Cedars-Sinai Medical Center, 8700 Beverly Blvd., Rm. 6732, Los Angeles, CA 90048.
Received 20 June 1995; accepted in final form 27 June 1996.
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