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Departments of Anesthesiology and of Physiology and Biophysics, Mayo Foundation, Rochester, Minnesota 55905
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
ABSTRACT 
Zhan, Wen-Zhi, Hirofumi Miyata, Y. S. Prakash, and Gary C. Sieck. Metabolic and phenotypic adaptations of diaphragm muscle
fibers with inactivation. J. Appl.
Physiol. 82(4):1145-1153, 1997.
We hypothesized
that metabolic adaptations to muscle inactivity are most pronounced
when neurotrophic influence is disrupted. In rat
diaphragm muscle
(Diam), 2 wk of
unilateral denervation or tetrodotoxin nerve blockade resulted in a
reduction in succinate dehydrogenase (SDH) activity of type I, IIa, and
IIx fibers (~50, 70, and 24%, respectively) and a decrease in SDH
variability among fibers (~63%). In contrast, inactivity induced by
spinal cord hemisection at C2 (ST)
resulted in much less change in SDH activity of type I and IIa fibers
(~27 and 24%, respectively) and only an ~30% reduction in SDH
variability among fibers. Actomyosin adenosinetriphosphatase (ATPase)
activities of type I, IIx, and IIb fibers in denervated and
tetrodotoxin-treated Diam were
reduced by ~20, 45, and 60%, respectively, and actomyosin ATPase
variability among fibers was ~60% lower. In contrast, only
actomyosin ATPase activity of type IIb fibers was reduced (~20%) in
ST Diam. These results suggest
that disruption of neurotrophic influence has a greater impact on
muscle fiber metabolic properties than inactivity per se.
spinal hemisection; tetrodotoxin; denervation; muscle fiber; succinate dehydrogenase activity; actomyosin adenosinetriphosphatase
activity
INTRODUCTION INNERVATION PLAYS an important role in establishing the
contractile and metabolic properties of muscle fibers (4). For example,
muscle fiber composition [myosin heavy chain (MHC) isoform expression] and enzymatic properties of motor units are
relatively homogeneous (12, 15, 16, 21, 22). In the cat diaphragm muscle (Diam), variability of
succinate dehydrogenase (SDH) activity among motor unit fibers is
comparable to that found along the length of individual fibers but
significantly less than that observed for adjacent fibers (22).
Moreover, motor unit fibers in the Diam usually express a single MHC
isoform (22). These results support the concept that the motoneuron
exerts a predominant control over the metabolic and phenotypic
properties of muscle fibers, which may occur either by the imposition
of a common activation history and/or by the influence of some
neurotrophic factor independent of activity.
In a recent study (14), we compared the impact of 2 wk of unilateral
Diam paralysis imposed in three
different ways: 1) unilateral
denervation (Dnv), where neurotrophic influence on muscle fibers was
completely removed; 2) tetrodotoxin
(TTX) blockade of phrenic nerve action potential propagation, where
phrenic motoneuron activity actually increased while the
Diam was paralyzed, causing a
mismatch between motoneuron and muscle activities that may have disrupted normal myoneural interactions; and
3) cervical spinal cord hemisection
at C2 [spinal transection
(ST)], where ipsilateral descending excitatory drive to phrenic
motoneurons from medullary premotor neurons was removed, causing both
phrenic motoneurons and the Diam
to become inactive. After 2 wk of unilateral
Diam paralysis induced by ST,
there was only a small decrease in muscle specific force [force
per cross-sectional area (CSA)] and no change in maximum unloaded
shortening velocity (14). In contrast, after Dnv or TTX blockade, there
was a marked decrease in Diam
specific force and a substantial slowing of maximum unloaded shortening velocity (14). Similar changes were also observed in the hamster Diam after Dnv and TTX blockade
(29). Therefore, we hypothesized that the effects of inactivity on
Diam mechanical properties are dependent on a match between phrenic motoneuron and muscle fiber activities, leading to intact and coherent myoneural interactions.
The purpose of the present study was to extend these previous
observations in the rat Diam by
comparing the adaptations in fiber MHC isoform expression, CSA, SDH
activity, and actomyosin adenosinetriphosphatase (ATPase) activity
induced by Dnv, TTX blockade, and ST. We hypothesized that, since
myoneural interactions are maintained in the ST model compared with the
Dnv and TTX models, Diam fiber
adaptations would be less pronounced in this model.
METHODS Male Sprague-Dawley rats (initial body weights 275 ± 6 g, 12 wk of
age) were randomly assigned to one of five groups:
1) untreated controls (Ctl;
n = 8);
2) surgical sham animals exposed to
anesthesia followed by laparotomy and neck incisions (Sham;
n = 5);
3) Dnv (n = 8);
4) TTX
(n = 8); and
5) ST
(n = 8). Animals were housed separately under a 12:12-h light-dark cycle and were provided Purina
rat chow and water ad libitum. All experimental
procedures were in strict accordance with the Animal Welfare Act.
Denervation. Surgical procedures were
performed while animals were anesthetized with ketamine (60 mg/kg) and
xylazine (2.5 mg/kg). The right phrenic nerve was transected in the
neck, and ~20 mm of distal nerve were removed to avoid reinnervation
and to minimize neurotrophic influence emanating from the remaining distal nerve stump (14, 28, 29).
TTX nerve blockade. A Silastic cuff,
connected to a miniosmotic pump (Alzet model 2002), was loosely placed
around the right phrenic nerve in the neck and sutured to surrounding
musculature. The nerve was then superfused with a 0.0125% saline
solution of TTX (pH 7.4) at a rate of 0.5 µl/h (14, 29).
Spinal hemisection. After dorsal
laminectomy, the right one-half of the spinal cord at
C2 was sectioned from the dorsal
to the ventral root. Care was taken to ensure that only the ventral and
lateral funiculi were cut and that blood vessels underneath the spinal
cord were avoided. This minimized motor deficits in the ipsilateral
limbs (14).
20°C (model 2800E Frigocut, Reichert-Jung). Alternate sections were stained for myofibrillar ATPase (mATPase) after preincubation at pH 4.3, 4.6, and 9.0, and at pH 10.4 after 4% paraformaldehyde fixation. Based on the mATPase staining profile, fibers were classified as type I, IIa, IIx, IIb, or IIc (3, 8, 24).
Five additional serial muscle sections were cut at 10 µm and reacted
with mouse primary antibodies against different MHC isoforms. In some
cases, only a single antibody was used, e.g.,
anti-MHCall-2X (Schiaffino BF-35).
However, in most cases, pairs of mouse immunoglobulin (Ig) G or IgM
primary antibodies were used, e.g.,
anti-MHCslow (Novocastra, IgG),
anti-MHC2A (Blau A4.74, IgG or
Blau N1.551, IgM), anti-MHC2B
(Schiaffino BFF3, IgM) and
anti-MHCNeo (Novocastra). Primary
antibodies were diluted in phosphate-buffered saline (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. The slides were then washed in
PBS and imaged with a Bio-Rad (MRC500/600) confocal system mounted on
an Olympus (BH2) microscope and equipped with an Ar-Kr laser.
Fiber SDH activity. Microscopic images
of muscle fibers were digitized at eight-bit resolution (256 gray
levels) into a 1,024 × 1,024 pixel array by using a video
image-processing system (MegaVision 1,024×M) that was calibrated
for densitometry and morphometry as previously described (2, 23, 24).
The quantitative histochemical procedure for measuring fiber SDH
activity has been previously described (2, 13, 23, 24). Briefly, the
reduction of nitroblue tetrazolium (NBT) to its diformazan (NBT-dfz)
was used as the reaction indicator. Four serial muscle sections were
cut at 6-µm thickness: two sections were reacted with
succinate added to the incubation medium and two sections
with succinate absent to control for nonspecific reduction of NBT. The
concentration of NBT-dfz deposited within a fiber during the SDH
reaction was determined from optical density (OD) measurements using
the Beer-Lambert equation
![[NBT-dfz] = OD/<IT>kl</IT>](/content/vol82/issue4/fulltext/1145/img001.gif)
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1 · cm1),
and l was the path length for light
absorbance (6 µm). From these measurements, the maximum velocity of
the SDH reaction was determined, and the mean SDH activity of each
fiber was expressed as millimoles fumarate per liter tissue per minute.
The SDH activities of ~125 fibers were analyzed in each muscle
sample.
Fiber actomyosin ATPase activity. The
quantitative histochemical procedure for measuring calcium-activated
actomyosin ATPase activity in muscle fibers has been previously
described (1, 24). Briefly, the amount of
Pi ions generated during the
hydrolysis of ATP was determined by measuring the concentration of a
lead sulfide precipitate (peak absorbance wavelength of 550 nm) by using the Lambert-Beer equation (see above), with a
k of 1,450 mol1 · cm1
for lead sulfide. The maximum velocity of the actomyosin ATPase reaction was determined and expressed as millimoles
Pi per liters tissue per minute.
The actomyosin ATPase activities of ~125 fibers were analyzed in each
muscle sample.
Statistical analysis. In preliminary
studies, we determined that a minimum sample of 20 fibers of each type
per muscle in six animals per experimental group was required to detect
a statistically significant (P < 0.05) change in each parameter from Ctl values at a 
level of 0.8. The effects of inactivity induced by the three models on
fiber SDH and actomyosin ATPase activities were evaluated by using a
two-way analysis of variance with fiber type and experimental group as
grouping variables. When appropriate, post hoc analyses were performed
by using a Student's t-test with Bonferroni correction. In previous studies, we found that fiber SDH
activities in the Diam are not
normally distributed (22-24). Therefore, to examine the effect of
inactivity on the variability of fiber SDH and actomyosin ATPase
activities, the interquartile range (IQR; range of measures around the
median accounting for 50% of all measurements) was calculated. The IQR
of each variable was calculated within a given fiber type and across
all fiber types as proportionately represented. Linear regression was
performed to determine correlations among fiber CSA, SDH activity, and
actomyosin ATPase activity. All data are presented as means ± SE.
RESULTS
Immediately after surgery, the animals lost weight for ~2 days, but subsequently body weight recovered in all experimental groups. Final body weights of all animals after 2 wk were comparable (335 ± 9 g).
Fiber type classification and MHC isoform
expression. In Ctl, Sham, and ST animals, the
histochemical classification of
Diam fiber types based on the pH
lability of mATPase staining corresponded with immunoreactivity for
different MHC antibodies (Fig. 1). In Dnv
and TTX animals, the histochemical classification of type I, IIa, IIx,
and IIb fibers also corresponded with immunoreactivity for
MHCslow,
MHC2A,
MHC2X, and
MHC2B isoforms, respectively.
However, the classification of type IIc fibers corresponded with the
coexpression of MHCslow and
MHC2A isoforms (Fig.
2). Based on immunoreactivity, it was not
possible to determine the relative expression of the MHCslow and
MHC2A isoforms in these type IIc
fibers, nor could the possible coexpression of the
MHC2X isoform be excluded. There was no detectable immunoreactivity against the
anti-MHCNeo in any of the
Diam sections
examined.
The proportions of different histochemical fiber types in the Diam of each group have been previously reported (14). Fiber type proportions determined based on MHC immunoreactivity differed only slightly from these previously reported values (Table 1). The proportions of different Diam fiber types were similar among Ctl, Sham, and ST groups. However, in Dnv and TTX animals, the proportions of fibers solely expressing MHC2A, MHC2X, and MHC2B isoforms were all significantly reduced compared with Ctl (Table 1; P < 0.05), whereas the proportion of fibers coexpressing MHCslow and MHC2A isoforms increased from <1% to ~30% (Table 1; P < 0.05).
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Fiber SDH activity. The SDH activity
of different fiber types in Ctl and Sham
Diam did not differ. In Ctl
Diam, there was a significant rank
order in SDH activities among the different fiber types, with
IIa>I>IIx>IIb (P < 0.05; Fig.
3). Compared with Ctl, SDH activities of
type I, IIa, and IIx fibers in Dnv and TTX
Diam decreased significantly
(P < 0.05; Fig. 3), whereas the SDH
activity of type IIb fibers did not change. The reduction in SDH
activities of type I and IIa fibers in Dnv and TTX
Diam was far more pronounced than
the decrease in type IIx fiber SDH activity
(P < 0.05; Fig. 3). In ST animals,
SDH activities of type I and IIa fibers also decreased compared with
Ctl (P < 0.05; Fig. 3) but to a
lesser extent than those observed in Dnv and TTX groups
(P < 0.05). The SDH activities of
type IIx and IIb fibers in the ST
Diam were not different from Ctl
values (Fig. 3). With these changes, SDH activities across fiber types
in the Dnv and TTX Diam were
relatively uniform (Fig. 3).
Table 2 summarizes the effect of 2 wk of inactivity on the variability of SDH activities among Diam fiber types. In the Ctl Diam, the IQR of type IIx fiber SDH activities was more than twofold greater than that for other fiber types (P < 0.05). With inactivity, the IQR of type IIx fiber SDH activities was reduced in all three models (P < 0.05) but to the greatest extent in the Dnv Diam (P < 0.05). The IQR of type I fiber SDH activities was reduced in Dnv and TTX Diam (P < 0.05) but was unaffected in ST Diam. In contrast, the IQR of type IIb fiber SDH activities increased in Dnv and TTX Diam (P < 0.05) but was unaffected in the ST animals. The IQR of type IIa fiber SDH activities was unaffected in all three models of inactivity. When all fiber types were combined proportionately, the IQR of SDH activities was significantly reduced after 2 wk of inactivity in all three models (P < 0.05) but to a greater extent in the Dnv and TTX Diam (P < 0.05).
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In Ctl Diam, smaller type I and
IIa fibers displayed higher SDH activities than larger type IIx and IIb
fibers (r2 = 0.70; P < 0.05; Fig.
4). In the Dnv and TTX animals, this
inverse correlation between fiber SDH activity and CSA disappeared (Dnv r2 = 0.18 and TTX
r2 = 0.19). In
contrast, an inverse correlation between fiber SDH activity and CSA
remained in the ST group
(r2 = 0.49;
P < 0.05; Fig. 4).
Fiber actomyosin ATPase activity. The
actomyosin ATPase activity of different fiber types in the Ctl and Sham
groups did not differ. In the Ctl
Diam, there was a significant rank
order in actomyosin ATPase activities among the different fiber types, with IIb>IIx>IIa>I (P < 0.05; Fig. 5). Compared with Ctl,
actomyosin ATPase activities of type I, IIx, and IIb fibers in Dnv and
TTX Diam decreased significantly
(P < 0.05; Fig. 5) but to a greater extent in type IIx and IIb fibers (P < 0.05). In the ST animals, the actomyosin ATPase activity of type
IIb fibers also decreased (P < 0.05;
Fig. 5) but to a much lesser extent than that observed in Dnv and TTX
groups (P < 0.05). The actomyosin
ATPase activities of type I, IIa, and IIx fibers in the ST
Diam were not different from Ctl
values (Fig. 5). The actomyosin ATPase activity of type IIa fibers was
also unaffected in all three experimental groups (Fig. 5). With these
changes in the Dnv and TTX Diam,
actomyosin ATPase activities across fiber types were relatively uniform
(Fig. 5).
Table 2 summarizes the effect of 2 wk of inactivity on the variability of actomyosin ATPase activities among Diam fiber types. In Ctl and ST animals, the IQR of Diam fiber actomyosin ATPase activities displayed a rank order with type IIb and IIx>IIa and I (P < 0.05). The IQR of type IIa fiber actomyosin ATPase activities was unaffected in all three models of inactivity. In Dnv Diam, the IQR of type I fiber actomyosin ATPase activities was lower compared with that observed in Ctl fibers (P < 0.05). In both Dnv and TTX animals, the IQR of type IIx and IIb fiber actomyosin ATPase activities was reduced compared with Ctl and ST Diam (P < 0.05). When all fiber types were combined proportionately, the IQR of actomyosin ATPase activity was reduced in the Dnv and TTX animals compared with both Ctl and ST animals (P < 0.05) but more so in the Dnv group (P < 0.05).
In Ctl Diam, smaller type I and
IIa fibers displayed lower actomyosin ATPase activities than larger
type IIx and IIb fibers (r2 = 0.47;
P < 0.05; Fig.
6). In the Dnv and TTX animals, this
correlation between fiber actomyosin ATPase activity and CSA
disappeared (Dnv r2 = 0.08 and TTX
r2 = 0.02). In
contrast, the correlation between fiber actomyosin ATPase activity and
CSA remained in the ST group
(r2 = 0.22;
P < 0.05; Fig. 6).
In Ctl animals, there was also a correlation between the actomyosin
ATPase and SDH activities of Diam
fibers (r2 = 0.43; P < 0.05; Fig.
7). After 2 wk of inactivity imposed by all
three models, the correlation between fiber actomyosin ATPase and SDH
activities disappeared
(r2 = 0.07, r2 = 0.02, and
r2 = 0.16 for
Dnv, TTX, and ST groups, respectively; Fig. 7).
DISCUSSION
The results of the present study demonstrate that morphological and metabolic adaptations of the Diam to inactivity are attenuated when myoneural interactions remain intact and coherent. These results are in agreement with those of our previous study, where it was shown that mechanical adaptations of the Diam were also attenuated in the ST model compared with both Dnv and TTX blockade (14). Therefore, it appears that Diam paralysis and/or muscle unloading per se are not the major factors contributing to the morphological, metabolic, and mechanical adaptations induced by Dnv and TTX blockade. Instead, the results suggest that a disruption of normal neurotrophic influence may play an important role in the adaptations. The similarity in adaptations induced by TTX and Dnv suggests that normal myoneural interactions depend on more than just intact innervation but also a match between motoneuron and muscle fiber activities. Based on the TTX model, it appears that continued motoneuron activity in the presence of muscle fiber inactivity leads to substantial change in muscle fiber morphological, metabolic, and mechanical properties.
Paralysis of the right Diam in each model was confirmed by the absence of EMG activity. In the ST model, the absence of EMG activity was also verified in the unanesthetized animal at different times during the 2-wk experimental period. During this time, the right phrenic motoneuron pool was also inactive. After TTX blockade, activity of the right phrenic nerve proximal to the block increased by ~50% (14), indicating that there was a compensatory increase in phrenic motoneuron activation. The nerve cuff and TTX superfusion did not cause any detectable nerve damage (14).
With unilateral paralysis of the Diam, it has been suggested that continued inspiratory-related contractions of the contralateral side may impose passive stretch on the inactivated hemidiaphragm, thereby affecting muscle adaptations (11, 25, 27). In a recent study in rabbits, we implanted sonomicrometry crystals into the midcostal and sternal regions of the right hemidiaphragm and examined the effects of unilateral Dnv on the passive strain imposed on these two regions by continued inspiratory-related activation of the contralateral side (28). After Dnv, the midcostal region displayed a small passive strain but insufficient to cause any measurable passive stress. In contrast, the sternal region of the Dnv Diam was passively shortened during inspiratory activation of the contralateral side. Both Diam regions showed similar morphological and mechanical adaptations after Dnv. Therefore, it appears that passive length changes and mechanical stress are not the main determinants of the morphological, metabolic, and mechanical adaptations induced by unilateral paralysis of the Diam.
In the present study, the effects of inactivity were studied only after 2 wk. In the rabbit Diam, hypertrophy of type I fibers and atrophy of type IIa and IIb fibers were observed after 1 wk of Dnv, and these morphometric changes became more pronounced after 4 wk (28). Talmadge and colleagues (26) reported that 15 days after complete transection of the midthoracic spinal cord in rats there were atrophy of the soleus muscle and alterations in MHC isoform expression. Furthermore, these investigators reported that by 30 days muscle adaptations induced by spinal cord transection were more pronounced. Therefore, it is clear that muscle adaptations in these different conditions of inactivity are dynamic, and it is possible that muscle remodeling may proceed at different rates depending on a variety of factors. In this regard, it should be noted that, although the adaptations in the ST Diam were attenuated in comparison with the Dnv and TTX groups, the changes in muscle fiber morphometry and metabolic properties were qualitatively similar in all three experimental groups. The Diam adaptations induced by ST differed from those observed in the rat soleus muscle after complete midthoracic spinal cord transection (26). Most notably, type I fibers hypertrophied in the Diam while type I fibers were reported to atrophy in the soleus. Specific muscle adaptations may relate to differences in the functional demands placed on the Diam and soleus. For example, ventilatory demands impose a rhythmic activation of the Diam with short periods of inspiratory activity (~400 ms) followed by short periods of inactivity associated with expiration (~500 ms). Thus, while the duty cycle is relatively high (~40%), the Diam does not display prolonged periods of activation. In contrast, locomotor and postural functions of the soleus muscle impose more prolonged periods of activity and inactivity. The mechanical loads placed on these two muscles during activity and inactivity are also quite different. The soleus muscle is a load-bearing muscle, whereas the Diam is not. The Diam also differs from the soleus muscle with regard to afferent feedback. The Diam has very few, if any, muscle spindles, whereas there are many muscle spindles in the soleus muscle. Accordingly, after ST, both phrenic motoneurons and the Diam were completely inactive, reflecting the absence of excitatory drive to phrenic motoneurons. This is not surprising given the fact that premotor neurons are located in the medulla. Furthermore, the major excitatory afferent influence on phrenic motoneurons derives from pulmonary stretch receptors, which have afferent axons in the vagus nerve and effects that are mediated through medullary neurons. In contrast, after thoracic spinal cord transection, it has been reported that there is continued residual activity in the soleus muscle. This continued activity of the soleus muscle after spinal cord transection, albeit reduced compared with normal animals, seriously complicates any interpretation of the underlying cause of muscle fiber adaptations. It also confuses any direct comparisons to the effects of inactivity on the Diam fibers.
Removal of neurotrophic influences emanating from the motoneuron may underlie the muscle adaptations after Dnv. In the rat extensor digitorum longus muscle, Dnv-induced atrophy was shown to be attenuated by injection of nerve extracts (6). In this study, the removal of neurotrophic influences by Dnv was reported to exert a more pronounced effect on the CSA of type IIb fibers compared with type IIa fibers (6). The atrophy of type IIb fibers was reversed by nerve extract, but the atrophy of type IIa fibers was not. Such a differential neurotrophic influence may explain the selective atrophy of type IIx and IIb fibers in the Diam after Dnv and TTX. A number of putative neurotrophic factors may be involved in maintaining normal morphological and metabolic properties of muscle fibers. For example, injection of ciliary neurotrophic factor has been shown to attenuate Dnv-induced atrophy of the rat soleus muscle (10). Previously, we reported that in the TTX model normal retrograde axonal transport of choleratoxin B fragment was present (14), suggesting that anterograde transport of neurotrophic factors also continued in phrenic nerves of TTX animals. Yet, muscle fiber adaptations were similar between the TTX and Dnv models. This observation suggests that removal of neurotrophic influence per se is not the primary factor affecting the muscle fiber adaptations or that the mismatch between motoneuron and muscle fiber activities in the TTX model somehow altered the normal neurotrophic influence.
After 2 wk of Diam paralysis induced by Dnv and TTX, there was a dramatic increase in the proportion of type IIc fibers. This is consistent with the results of Gauthier and Hobbs (7), who reported an increased proportion of type IIc fibers in the rat Diam after 4 wk of unilateral Dnv. The type IIc fibers in the Dnv and TTX Diam coexpressed the MHCslow and MHC2A isoforms. This observation is also consistent with other previous reports showing coexpression of slow and fast MHC isoforms in type IIc fibers of the rat Diam after Dnv (5, 7). In rat hindlimb muscles, Schiaffino et al. (20) reported that 3-7 days after Dnv there was reexpression of the MHCNeo isoform. In the Dnv Diam, reexpression of the MHCNeo isoform was also found, but it comprisied <1% of the total MHC. As early as 1-3 days after unilateral Dnv, there is increased mitotic activity of satellite cells in the rat Diam (9). It is possible that satellite cell activation may be an important factor initiating coexpression of MHC isoforms within single Diam fibers.
The expression of different MHC isoforms is associated with differences in cross-bridge cycling rates and maximum shortening velocity of muscle fibers (18, 19). In the Dnv and TTX Diam, the coexpression of MHCslow and MHC2A isoforms in type IIc fibers may affect a mismatch of cross-bridge cycling rates within these fibers and present an internal load to shortening (18). Such a possibility is consistent with the observed reduction in maximum shortening velocity of the Dnv and TTX Diam (14).
In a previous study (9), we found that satellite cell activation in the denervated rat Diam was associated with an increased incidence of muscle fiber injury, possibly as a result of microfasciculations. Although the extent of muscle fiber injury in each model was not systematically evaluated in the present study, it is possible that injury was less prevalent in the ST Diam and that such differences could underlie variations in the degree of muscle remodeling.
After 2 wk of Diam inactivity imposed by all three models, there was a decrease in the SDH activity of both type I and IIa fibers. This indicates that activation history is an important determinant of mitochondrial function in muscle fibers. It is of interest that the reduction in SDH activity in type I and IIa fibers in the ST Diam was less pronounced than that observed in the Dnv and TTX animals. It is possible that the hypertrophy of type I and IIa fibers in the Dnv and TTX Diam exaggerated the reduction in SDH activity of these fibers. In this context, the SDH activity of type IIx fibers in the Dnv and TTX Diam decreased despite the atrophy of these fibers.
The actomyosin ATPase activity of type IIx and IIb fibers in the Dnv and TTX Diam decreased. It is doubtful that inactivity per se was the underlying cause of the reduction of actomyosin ATPase activity in these fibers. The actomyosin ATPase activity of type I fibers was also reduced in the Dnv and TTX Diam. Overall, the lower actomyosin ATPase activity of fibers in the Dnv and TTX Diam corresponds to the slower shortening velocity in these muscle (14). In the ST Diam, only the actomyosin ATPase activity of type IIb fibers was reduced but to a much lesser extent, compared with that observed in the Dnv and TTX animals. The overall maintainance of fiber actomyosin ATPase activity in the ST Diam corresponds to the lack of any change in shortening velocity in this muscle (14).
Both Dnv and TTX reduced the variability of CSA, SDH activity, and actomyosin ATPase activity across different Diam fiber types. In the cat Diam, we found that the variability of SDH activity among muscle fibers belonging to single-motor units was comparable to that found along the length of individual muscle fibers and far less than that found across all fibers (22). A similar homogeneity of enzymatic properties among motor unit fibers has been reported by other investigators (15, 16), leading to the conclusion that motoneurons exert a predominant control over the metabolic properties of muscle fibers (22). The results of the present study suggest that the influence exerted by a motoneuron occurs, to a limited extent, by the imposition of a common activation history on motor unit fibers but mostly by the influence of some neurotrophic factor independent of activity.
In conclusion, the results of the present study indicate that adaptations in morphological and metabolic properties of the rat Diam after inactivity are significantly attenuated if a match between phrenic motoneuron and muscle fiber activity is maintained. It is likely that the marked adaptations in the Dnv and TTX Diam were not due to muscle paralysis per se but to alterations in myoneural interactions. In the Dnv model, neurotrophic influence was completely removed and, in the TTX model, the mismatch between motoneuron activity and muscle fiber inactivity most likely altered normal neurotrophic influences. The similarity of the morphological and metabolic adaptations induced by TTX and Dnv and the general lack of adaptation in the ST Diam lead us to conclude that when there is a match between phrenic motoneuron and muscle fiber activities and, therefore, myoneural interactions remain intact and coherent, normal neurotrophic influences are preserved, and muscle fiber adaptations to inactivity are attenuated.
ACKNOWLEDGEMENTS
The authors express their thanks to Yun Hua Fang for her assistance in the histochemical analyses.
FOOTNOTES
Address for reprint requests: G. C. Sieck, Anesthesia Research, Mayo Clinic, Rochester, MN 55905 (E-mail: gcs{at}siecklab.mayo.edu).
Received 27 June 1996; accepted in final form 20 November 1996.
REFERENCES
| 1. | Blanco, C. E., and G. C. Sieck. Quantitative determination of calcium-activated myosin adenosine triphosphatase activity in rat skeletal muscle fibres. Histochem. J. 24: 431-444, 1992. [Medline] . |
| 2. | Blanco, C. E., G. C. Sieck, and V. R. Edgerton. Quantitative histochemical determination of succinic dehydrogenase activity in skeletal muscle fibres. Histochem. J. 20: 230-243, 1988. [Medline] . |
| 3. | Brooke, M. H., and K. K. Kaiser. Three "myosin adenosine triphosphatase" systems: the nature of their pH lability and sulfhydryl dependence. J. Histochem. Cytochem. 18: 670-672, 1970. [Medline] . |
| 4. | Buller, A. J., J. C. Eccles, and R. M. Eccles. Differentiation of fast and slow muscles in the cat hind limb. J. Physiol. (Lond.) 150: 399-416, 1960. . |
| 5. |
Carraro, U.,
D. Morale,
I. Mussini,
S. Lucke,
M. Cantini,
R. Betto,
C. Catani,
L. D. Libera,
D. D. Betto,
and
D. Noventa.
Chronic denervation of rat hemidiaphragm: maintenance of fiber heterogeneity with associated increasing uniformity of myosin isoforms.
J. Cell Biol.
100:
161-174,
1985.
|
| 6. | Davis, H. L., and J. A. Kiernan. Effect of nerve extract on atrophy of denervated or immobilized muscle. Exp. Neurol. 72: 582-591, 1981. [Medline] . |
| 7. | Gauthier, G. F., and A. W. Hobbs. Effects of denervation on the distribution of myosin isozymes in skeletal muscle fibers. Exp. Neurol. 76: 331-346, 1982. [Medline] . |
| 8. | Gorza, L. Identification of a novel type 2 fiber population in mammalian skeletal muscle by combined use of histochemical myosin ATPase and anti-myosin monoclonal antibodies. J. Histochem. Cytochem. 38: 257-265, 1990. [Abstract] . |
| 9. |
Gosselin, L. E.,
G. Brice,
B. Carlson,
Y. S. Prakash,
and
G. C. Sieck.
Changes in satellite cell mitotic activity during the acute period of unilateral diaphragm denervation.
J. Appl. Physiol.
77:
1128-1134,
1994.
|
| 10. | Helgren, M. E., S. P. Squinto, H. L. Davis, D. J. Parry, T. G. Boulton, C. S. Heck, Y. Zhu, G. D. Yancopoulos, R. M. Lindsay, and P. S. DiStefano. Trophic effect of ciliary neurotrophic factor on denervated skeletal muscle. Cell 76: 493-504, 1994. [Medline] . |
| 11. | Hopkins, D., K. L. Manchester, and M. Gregory. Histochemical and biochemical characteristics of the transient hypertrophy of the denervated rat hemidiaphragm. Exp. Neurol. 81: 279-293, 1983. [Medline] . |
| 12. |
Larsson, L.,
L. Edstrom,
B. Lindegren,
L. Gorza,
and
S. Schiaffino.
MHC composition and enzyme-histochemical and physiological properties of a novel fast-twitch motor unit type.
Am. J. Physiol.
261 (Cell Physiol. 30):
C93-C101,
1991.
|
| 13. | Martin, T. P., A. C. Vailas, J. B. Durivage, V. R. Edgerton, and K. R. Castleman. Quantitative histochemical determination of muscle enzymes: biochemical verification. J. Histochem. Cytochem. 33: 1053-1059, 1985. [Abstract] . |
| 14. |
Miyata, H.,
W. Z. Zhan,
Y. S. Prakash,
and
G. C. Sieck.
Myoneural interactions affect muscle adaptations to inactivity.
J. Appl. Physiol.
79:
1640-1649,
1995.
|
| 15. |
Nemeth, P. M.,
D. Pette,
and
G. Vrbova.
Comparison of enzyme activities among single muscle fibres within defined motor units.
J. Physiol. (Lond.)
311:
489-495,
1981.
|
| 16. | Nemeth, P. M., L. Solanki, D. A. Gordon, T. M. Hamm, R. M. Reinking, and D. G. Stuart. Uniformity of metabolic enzymes within individual motor units. J. Neurosci. 6: 892-898, 1986. [Abstract] . |
| 17. | Prakash, Y. S., M. Fournier, and G. C. Sieck. Effects of prenatal undernutrition on developing rat diaphragm. J. Appl. Physiol. 74: 1044-1052, 1993. . |
| 18. |
Reiser, P. J.,
R. L. Moss,
G. G. Giulian,
and
M. L. Greaser.
Shortening velocity and myosin heavy chains of developing rabbit muscle fibers.
J. Biol. Chem.
260:
14403-14405,
1985.
|
| 19. | Schiaffino, S., S. Ausoni, L. Gorza, L. Soggin, K. Gundersen, and T. Lomo. Myosin heavy chain isoforms and velocity of shortening of type 2 skeletal muscle fibres. Acta Physiol. Scand. 134: 575-576, 1988. [Medline] . |
| 20. | Schiaffino, S., L. Gorza, G. Pitton, L. Saggin, S. Ausoni, S. Sartore, and T. Lomo. Embryonic and neonatal myosin heavy chain in denervated and paralyzed rat skeletal muscle. Dev. Biol. 127: 1-11, 1988. [Medline] . |
| 21. | Sieck, G. C., M. Fournier, and J. G. Enad. Fiber type composition of muscle units in the cat diaphragm. Neurosci. Lett. 97: 29-34, 1989. [Medline] . |
| 22. |
Sieck, G. C.,
M. Fournier,
Y. S. Prakash,
and
C. E. Blanco.
Myosin phenotype and SDH enzyme variability among motor unit fibers.
J. Appl. Physiol.
80:
2179-2189,
1996.
|
| 23. |
Sieck, G. C.,
R. D. Sacks,
C. E. Blanco,
and
V. R. Edgerton.
SDH activity and cross-sectional area of muscle fibers in cat diaphragm.
J. Appl. Physiol.
60:
1284-1292,
1986.
|
| 24. |
Sieck, G. C.,
W. Z. Zhan,
Y. S. Prakash,
M. J. Daood,
and
J. F. Watchko.
SDH and actomyosin ATPase activities of different fiber types in the rat diaphragm muscle.
J. Appl. Physiol.
79:
1629-1639,
1995.
|
| 25. | Sola, O. M., and A. W. Martin. Denervation hypertrophy and atrophy of the hemidiaphragm of the rat. Am. J. Physiol. 172: 324-332, 1953. . |
| 26. |
Talmadge, R. J.,
R. R. Roy,
and
V. R. Edgerton.
Prominence of myosin heavy chain hybrid fibers in soleus muscle of spinal cord-transected rats.
J. Appl. Physiol.
78:
1256-1265,
1995.
|
| 27. | Yellin, H. Changes in fiber types of the hypertrophying denervated hemidiaphragm. Exp. Neurol. 42: 412-428, 1974. [Medline] . |
| 28. |
Zhan, W. Z.,
G. A. Farkas,
M. A. Schroeder,
L. E. Gosselin,
and
G. C. Sieck.
Regional adaptations of rabbit diaphragm muscle fibers to unilateral denervation.
J. Appl. Physiol.
79:
941-950,
1995.
|
| 29. |
Zhan, W. Z.,
and
G. C. Sieck.
Adaptations of diaphragm and medial gastrocnemius muscles to inactivity.
J. Appl. Physiol.
72:
1445-1453,
1992.
|
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