1 Institut National de la
Santé et de la Recherche Médicale (INSERM) Unité 460 and 4 Services de Pneumologie des
Hôpitaux Bichat et Beaujon et INSERM Unité 408, In striated muscle, chronic increases in
workload result in changes in myosin phenotype. The aim of this study
was to determine whether such changes occur in the diaphragm of
patients with severe chronic obstructive pulmonary disease, a situation
characterized by a chronic increase in respiratory load and lung
volume. Diaphragm biopsies were obtained from 22 patients who underwent
thoracic surgery. Myosin was characterized with electrophoresis in
nondenaturing conditions, SDS-glycerol PAGE, and Western blotting with
monoclonal antibodies specific for slow and fast myosin heavy chain
(MHC) isoforms. Flow volume curves, total lung capacity, and functional residual capacity were measured before surgery in 20 patients. We found
that the human diaphragm is composed of at least four myosin isoforms,
one slow and three fast, resulting from the combination of three MHC
species. Chronic overload was associated with an increase in the slow
human diaphragm; myosin electrophoresis; chronic obstructive
pulmonary disease; lung distension
CHRONIC OBSTRUCTIVE PULMONARY DISEASE (COPD) is
characterized by increased resistance to airflow, air trapping, and
hyperinflation of the lungs. The increased resistance to airflow
increases the work and energy required for breathing. Furthermore, lung
hyperinflation places the inspiratory muscles, and particularly the
diaphragm, at a mechanical disadvantage. Because most of the
ventilatory burden in these patients is borne by the inspiratory
muscles, the muscles, and especially the diaphragm, can fatigue and
fail as pressure generators, leading to respiratory failure. Indeed, there is now ample evidence that the inspiratory muscles may fail if
chronically overloaded (16). Inspiratory muscle fatigue has been
documented in acute ventilatory failure and in chronic hypercapnic COPD
when ventilation is voluntarily increased (19). However, in patients
with stable severe COPD, the inspiratory muscles are capable of coping
chronically with very high loads. Moreover, it has been shown recently
in well-nourished patients with lung hyperinflation and stable COPD
that the strength of diaphragm contraction and, to a greater extent,
its inspiratory action are well preserved (27). This suggests that, in
such patients, changes occur in the structure and function of the
muscle that allow it to adapt to the altered functional demands.
Such adaptive alterations in the structure and function of striated
muscles, including the heart, have been widely documented in a number
of physiological and pathological conditions (for a review, see Ref.
30). With respect to skeletal muscle, it has been shown that chronic
stimulation of a fast-twitch muscle at a slow rate results in a number
of structural and functional changes that affect not only contractile
protein phenotype but also capillary bed density, function of the
sarcoplasmic reticulum, and the enzymes of both aerobic and anaerobic
metabolism, leading to a fatigue-resistant, slowly contracting muscle
(30). Among the changes in contractile protein phenotype, those of
myosin heavy chain (MHC) are of special physiological importance
because MHC is mainly responsible for the level of ATPase activity of the whole myosin molecule, which itself correlates with the maximum shortening velocity of the muscle fiber (3, 25, 30). During the last
two decades, our knowledge of changes in the myosin phenotype of
striated muscles during a variety of physiological and pathological conditions has improved greatly through the use of discriminatory electrophoretic techniques such as electrophoresis in nondenaturing conditions, which allows myosin forms to be separated according to
charge differences of the whole hexameric molecule (10), and original
SDS-PAGE, which can separate the various MHC isoforms (2, 5). Together
with histochemistry, these techniques have shown that, after a chronic
increase in muscle load, a shift occurs in myosin isoforms, from those
with high ATPase activity (composed of "fast" MHCs) to those
responsible for low ATPase activity (composed of Little is known about diaphragmatic myosin phenotypes or possible
alterations during pathological situations. In the diaphragm of the
adult rat, five myosin bands (one slow- and four fast-migrating bands)
have been detected by pyrophosphate gel electrophoresis (13). The four
fast-migrating adult isoforms gradually replace embryonic and neonatal
isoforms, whereas the amount of the slow isoform increases. Using a
specific PAGE method with 30% glycerol, LaFramboise et al. (14) were
able to isolate four MHC isoforms from the diaphragm of the adult rat:
type I ( Patients, measurement of ventilatory function, and
muscle sampling. We studied 22 patients (18 men and 4 women with a mean age of 54.4 ± 8.7 yr) due for thoracic surgery
for lung cancer or resection of a large bulla (Table
1). None of them was receiving chronic glucocorticoid administration, and all had normal nutritional status. Ventilatory function was thoroughly documented before surgery
(Table 2) except in two
patients with lung metastases whose respiratory function was normal on
a flow-volume curve. All measurements were performed in the sitting
position. Several flow-volume curves were obtained with a
Hewlett-Packard spirometer (Hewlett-Packard, Waltham, MA) to determine
the forced expiratory volume in 1 s
(FEV1) and forced vital
capacity. Total lung capacity (TLC) and functional residual capacity
(FRC) were measured with a constant-volume body plethysmograph (Gould
System 2800; Gould Instruments, Cleveland, OH). Muscle biopsy specimens
of ~50-100 mg were obtained during surgery, in keeping with our
institutional guidelines for human research, in the same costal region
of the diaphragm. In addition, a sample of the pectoralis major was
obtained from a patient undergoing a radical mastectomy. All muscle
specimens were placed in liquid nitrogen immediately after sampling and stored at
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
-MHC species at the expense of the fast species (
-MHC, 78.2 ± 4.6 and 50.0 ± 6.5% in emphysematous and control patients,
respectively; P < 0.005). Linear
correlations were found between
-MHC percentage and forced
expiratory volume in 1 s (r =
0.52;
P < 0.02), total lung capacity
(r = 0.44;
P < 0.05), and functional residual
capacity (r = 0.65;
P < 0.003). The human adult
diaphragm is composed of a balanced proportion of slow and fast myosin
isoforms. In patients with chronic obstructive pulmonary disease, the
proportion of fast myosins decreases, whereas that of slow myosin
increases. This increase appears to be closely related to lung
hyperinflation and may reflect an adaptation of the diaphragm to the
new functional requirements.
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
-MHC) (21, 30). It
has also been clearly established that this myosin transition is
responsible, at least in part, for the decreased shortening velocity of
the unloaded muscle and for an improvement in the economy of force
generation (1, 21, 26, 30).
/slow) MHC, type IIa and IIx MHCs (present in approximately
equal proportions in both the crural and costal portions of the
muscle), and type IIb MHC, a minor isoform. To our knowledge, no data
have been published on the human diaphragmatic MHC phenotype or changes
in response to chronic increases in respiratory load and lung volume.
The aim of this study was therefore to establish the MHC phenotype of
the human diaphragm and to identify any changes in patients with severe
stable COPD. We also investigated possible correlations of the MHC
phenotype with indexes of respiratory function, which account for the
severity of airway obstruction and lung hyperinflation.
METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
80°C until use.
Table 1.
Patient profiles
Table 2.
Ventilatory function and slow myosin percentage
Gel electrophoresis in nondissociating
conditions. About 20 mg of the muscle sample were
crushed in liquid nitrogen and extracted at 4°C for 20 min with 4 volumes (wt/vol) of a slightly modified Guba's solution [0.3 M
KCl, 0.1 M
H2KPO4,
0.05 M
HK2PO4,
0.001 M MgCl2, 0.01 M
Na4P2O7,
1% (wt/vol) Na azide, and 1% (vol/vol) 2-mercaptoethanol] as
previously described (10, 18). The homogenate was centrifuged at
4°C and 30,000 g for 20 min, and
the supernatent was stored at 20°C in 50% (vol/vol)
glycerol. PAGE in nondissociating conditions was performed with a
Pharmacia apparatus (GE-4II) capable of thermostating and circulating
the buffer between the anodic and cathodic reservoirs (10, 18).
Briefly, cylindrical polyacrylamide gels (60 × 5 mm) were
prepared with 3.88% (wt/vol) acrylamide-0.12% (wt/vol)
N,N'-methylene-bis-acrylamide.
Each gel was loaded with 30 µl of a 40-fold dilution of the crude
muscle extract (1-2 mg of myosin) with 0.01 M
Na4P2O7,
50% (vol/vol) glycerol, and traces of bromphenol blue at pH 8.5. The
buffer was kept at between 1 and 3°C, and electrophoresis was run
at a constant voltage of 14 V/cm for 20-24 h. Staining and
destaining of the gels were carried out in a Hoefer Scientific
Instruments apparatus. Densitometric tracings of the gels were obtained
with a Gilford model 240 spectrophotometer equipped with a
Hewlett-Packard 70-44A multirecorder tracing table. The relative
amounts of slow and fast myosin isoforms were calculated from the
height of each peak. The reproducibility of the method has been shown
to be ~9% (18).
SDS-PAGE and Western blotting. Myosin was extracted by using a rapid procedure described by Schiaffino et al. (23). Briefly, muscle fragments were homogenized in 10 volumes of 20 mM KCl-2 mM K2HPO4-1 mM EGTA, pH 6.8, and centrifuged at 12,000 g for 2 min. After a wash with the same buffer, the pellets were resuspended in 40 mM Na4P2O7-1 mM MgCl2-1 mM EGTA, pH 9.5, for 15 min to extract myosin. The samples were then centrifuged at 12,000 g for 15 min, and the protein concentration in the supernatant was determined according to Bradford's method (Bio-Rad). Myosin electrophoresis was performed using 6% polyacrylamide gels in the presence of 37.5% glycerol (24). Similar amounts of MHC, as determined by densitometry, were loaded on each lane.
Three monoclonal antibodies were used for the immunochemical
characterization of MHCs. The first, BA-D5, was directed against /slow MHC; the second, SC-75, against type II MHCs; and the third, BF-32, against both
/slow and type IIa MHCs. We call type IIa the
fast MHC isoform recognized by BF-32 because only type I and IIa fibers
in human muscle sections were labeled by this antibody, type IIb fibers
being unreactive (data not shown). The procedure described by Towbin et
al. (32) was used for Western blotting experiments. Briefly, proteins
separated on SDS-polyacrylamide gels were transfered onto
nitrocellulose paper at 250 mA overnight in 25 mM
Tris · HCl-192 mM glycine buffer, pH 8.00. The paper sheets were incubated with appropriate dilutions of monoclonal antibodies, and reactivity was revealed by immunoperoxidase staining with rabbit anti-mouse IgG conjugated with horseradish peroxidase (Dako, Milan, Italy); diaminobenzidine (Serva, Milan, Italy) was used
as the substrate in the presence of imidazole (22). The gels stained
with Coomassie blue were scanned with a Shimadzu CS-930 dual-wavelength
thin-layer chromatography scanner to estimate the relative
amounts of the MHC bands.
Expression of the results and statistical
analysis. For quantitative analysis, only the bands
corresponding to slow myosin and -MHC were considered
in pyrophosphate and SDS-polyacrylamide gel analysis, respectively. The
proportions of slow myosin and
-MHC are expressed as the percentage
of the corresponding forms among total myosin and MHCs, respectively.
The nonparametric Mann-Whitney test was used for group comparisons and
regression analysis. P values of
<0.05 were considered to denote significant differences.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Myosin electrophoresis in nondenaturing
conditions. Figure 1 shows
typical electrophoretic patterns of native diaphragmatic myosin from
three patients undergoing bulla resection [emphysematous patients
(patients E1-3)]
or lung cancer surgery [control patients ( patients C1-3)].
In these electrophoretic conditions, myosin from type I
fatigue-resistant muscle fibers (myosin composed of -MHC) is also
the one that exhibits the slowest electrophoretical mobility. Like
diaphragmatic myosin in other mammalian species, diaphragmatic myosin
in the two categories of patients separated into fast- and
slow-migrating bands corresponding to fast and slow myosin isoforms,
respectively. However, the relative amounts of fast and slow myosin
forms differed markedly from patient to patient in both groups. Slow
myosin was detected in all samples as a single dark band or a major
dark band associated with a faint band migrating slightly ahead. In
sharp contrast, the abundance of fast myosin isoforms differed greatly
among the patients. Three fast myosin bands were clearly detected in
some patients ( patients E1 and
C1), whereas fast myosins were at
the detection limit in others ( patients
E3 and C3). An
intermediate pattern was observed in patients
E2 and C2. Table 2
shows the proportions of slow myosin relative to total myosin. Because
the faint band migrating sightly ahead of the major slow myosin band
appeared as a small shoulder of the major peak on densitometric
tracings, the proportion of slow myosin among total myosin (slow myosin
percentage) was estimated only from the height of the major peak.
|
MHC SDS-PAGE. Diaphragmatic myosin
from 8 of the 22 patients was submitted to SDS-PAGE and MHC
immunochemical characterization. As with native myosin electrophoresis,
SDS-PAGE revealed both the multiplicity of MHC isoforms and marked
differences in the electrophoretic pattern among patients. Figure
2A shows
examples of the different electrophoretic patterns in four patients. In contrast to pyrophosphate gel electrophoresis, the fast-migrating band
corresponds, in both human and rat diaphragms, to myosin composed of
type I (/slow) MHCs, whereas the slow-migrating bands correspond to
myosins composed of fast MHCs. One fast- and two slow-migrating bands
were detected in two of the four samples ( patients C4 and
C5), probably corresponding, by
analogy with other muscles and mammalian species, to slow (type I) and
fast (type II) MHCs, respectively. Type I and II MHCs were present in
approximately equal proportions, and among the two type II MHC bands,
the faster migrating species predominated. Type I myosin was
consistently observed in all the samples studied, whereas the
proportion of type II MHCs varied markedly, being equivalent to that of
type I MHC in some samples ( patients
C4 and C5), almost undetectable in another ( patient
E4), and intermediate in the remainder
( patient E3). In the latter
case, the faster migrating type II MHC species always predominated and
was sometimes the only visible MHC form. Figure
2B shows the electrophoretic patterns of myosin in nondenaturing conditions. In the eight muscle samples subjected to both denaturing and nondenaturing electrophoresis, there
was a strong link (r = 0.97;
P < 0.0001) between the proportion of slow myosin (Fig. 2B, S) and type I
MHC (Fig. 2A, I).
|
Characterization of MHCs by Western blot
analysis. Figure
3A shows
the electrophoretic pattern (Coomassie blue staining) of myosin
purified from three patients ( patients
C5, C4, and
E4) and from a human adult pectoral
muscle (P). Three MHC bands were separated in the pectoral muscle and
in two diaphragmatic samples ( patients
C5 and C4) and
corresponded to type I, IIa, and IIb MHCs, respectively, based on the
nomenclature used by Klitgaard et al. (12). The electrophoretic pattern
of myosin from the diaphragm of patients
C5 and C4 was
identical to that of myosin from the pectoral muscle. The first
monoclonal antibody, BA-D5, reacted against the fast-migrating band in
all four samples, confirming that this band was composed of type I
(/slow) MHC (Fig. 3B). The second
monoclonal antibody, SC-75, reacted with the two slow-migrating MHC
bands in patients C5 and
C4 and the pectoral muscle and with a
band in patient E4, migrating the same
distance as the faster component of the two slow-migrating bands in
patients C5 and
C4 and the pectoral muscle, confirming
that these slow-migrating bands were composed of type II MHCs (Fig.
3C). The third monoclonal antibody,
BF-32, which labeled type I and IIa but not type IIb fibers in human
muscle sections, reacted with both the fast-migrating band and the fast
component of the slow-migrating bands, indicating that these bands were
composed of type I (
/slow) and type IIa MHCs, respectively (Fig.
3D). Finally, the sample from
patient E4 contained an increased
proportion of type I (
/slow) MHC and a decreased proportion of type
IIa MHC relative to patients C4 and
C5, whereas type IIb MHC was
undetectable.
|
Slow myosin in the diaphragm of patients with and
without severe COPD and correlations with ventilatory
function. Analysis of native myosin phenotype in the 20 patients in whom ventilatory function was assessed allowed us to
establish that the proportion of slow myosin among total myosin was
increased by 56% in those patients with severe stable COPD compared
with control patients (78.2 ± 4.6 vs. 50.0 ± 6.5%;
P < 0.005; Table 2). Grouping
patients according to their functional capacity rather than their
diagnosis would lead to a very similar result as seen from the values
in Table 2. In addition, we could identify correlations between the
proportion of slow myosin and parameters reflecting a chronic increase
in airway resistance (FEV1) or
lung distension (TLC and FRC) in all study patients except two
( patients C3 and
C4) with lung metastases whose
respiratory function was normal on a flow-volume curve. The raw view of
the data scattergram was highly suggestive for linear correlations
between the above-mentioned variables, which was confirmed by linear
regression analysis. Indeed, we found a negative linear
correlation between
FEV1 and the slow myosin
percentage (r = 0.52;
P < 0.02) and positive linear
correlations between the slow myosin percentage and both TLC and FRC
(r = 0.44; P < 0.05 and
r = 0.65;
P < 0.003, respectively; Fig.
4). Because these results
confirmed our initial assumption that myosin phenotypic conversion
toward type I (
/slow) MHC is proportional to lung distension and
airway obstruction, we did not test whether the variables were related
in a nonlinear way. The best correlation coefficient
(r = 0.65;
P < 0.003) was observed between slow
myosin percentage and FRC, suggesting that the position of the
diaphragm at the end of spontaneous expiration may be an important
determinant of MHC phenotypic change in patients with COPD.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
These results show that the human diaphragm is composed of at least
four myosin species resulting from the combination of three MHC
isoforms and that chronic mechanical overload of the human diaphragm
results in the accumulation of slow -MHC at the expense of the fast
isoforms. In addition, our results suggest that this MHC transition
appears to increase with the deterioration of respiratory function
(i.e., airway obstruction and especially lung hyperinflation).
Pyrophosphate gel analysis separates myosin isoforms according to charge differences of the whole hexameric myosin molecule. This technique allowed us to identify two band groups in crude protein extracts from the human diaphragm: the first one was a fast-migrating group composed of three bands and the second was a slow-migrating group composed of, in most cases, a dark major band associated with a faint band migrating slightly ahead (some patients had a single dark band). By analogy with a variety of muscles from various mammalian species, these two band groups probably corresponded to fast and slow myosin isoforms, respectively (13, 14, 34). Analysis of necropsy samples from various areas of the costal diaphragm from a patient free of bronchopulmonary disease (data not shown) indicated that this band did not arise from postmortem myosin proteolysis and showed no significant difference in the myosin pattern throughout the normal human costal diaphragm.
The proportion of slow myosin is smaller in the diaphragm of the rat (13, 14) than in the patients studied here whose respiratory function was normal or close to normal. Because the ventilatory rate in the former species is higher than in humans, the myosin phenotype of diaphragm muscle may be related to it. This is reminiscent of the differences in cardiac myosin phenotype among mammalian species and of the relationships among the proportion of a given myosin species, myosin ATPase activity, and the maximum contraction velocity of the muscle, which have to be compatible with heart rate (6, 15). In the ventricles of small mammals, fast myosin, high myosin ATPase activity, and contraction velocity seem to be adapted to a high heart rate but at the expense of the efficiency of muscle contraction (1, 26). Conversely, in the ventricles of big mammalian species, the almost exclusive expression of slow myosin (9, 15) appears adapted to low heart rates and may be regarded as an adaptive evolutionary mechanism. In this respect, the larger proportion of slow myosin in the human diaphragm than in that of small rodents could be also interpreted as an evolutionary adaptation of the human diaphragm to a lower ventilatory rate.
PAGE in the presence of SDS and high glycerol concentrations can be
used to separate MHC isoforms. In contrast to pyrophosphate gel
electrophoresis, the fast-migrating band corresponds, in both human and
rat diaphragms, to myosin composed of type I (/slow) MHCs, whereas
the slow-migrating bands correspond to myosins composed of fast MHCs.
The fact that the fast-migrating MHC isoform accounted for
approximately one-half of the total MHC in samples from patients with
normal or close-to-normal respiratory function is in keeping with the
proportion of the slow-migrating band in pyrophosphate gel analysis of
the corresponding muscle samples and confirms that the normal human
diaphragm contains a balanced proportion of fast and slow MHCs in total
myosin. Among the 20 patients whose diaphragmatic samples were
subjected to the two electrophoretic procedures, we observed a strong
positive linear correlation between the proportion of slow myosin in
pyrophosphate gel electrophoresis and the proportion of type I
(
/slow) MHC in SDS-glycerol PAGE, confirming that the slow-migrating
band observed with the former technique corresponds to myosin composed
of
-MHCs. This was further borne out by Western blot analysis
because the fast-migrating MHC band reacted with monoclonal antibodies
(BA-D5 and BF-32) directed against
/slow MHCs and type I and IIa
fibers in human muscle sections, respectively.
In contrast to /slow MHC (fast-migrating band), SDS-glycerol PAGE
and immunochemical analysis revealed marked differences in the pattern
of fast MHCs (slow-migrating bands) between human and rat diaphragms.
First, only two bands were detected in the human diaphragm compared
with three in the rat diaphragm (14). This is due to the presence in
the latter species of an additional band corresponding to an additional
MHC, type IIx, which migrates between the two type II (a and b) MHCs
(14). The two bands detected in the human diaphragm were type II MHCs
because they both reacted with monoclonal antibody SC-75. In addition,
monoclonal antibody BF-32 showed that the fastest migrating MHC
component in the human diaphragm was type IIa, whereas the fastest
migrating band in the rat diaphragm was type IIb (14). The low-mobility
MHC band in human muscle was initially identified as type IIb (4, 12), but in situ hybridization combined with ATPase histochemistry and
anti-MHC immunocytochemistry on serial sections (28) later indicated
that human muscle fibers identified as type IIb (based on ATPase
histochemistry) contained MHC transcripts homologous to rat type IIx
MHC transcripts (7). On the other hand, type I and type IIa human
muscle fibers contain transcripts homologous to rat
/slow and type
IIa MHCs, respectively (28). The presence of type IIx-like mRNAs in
human type IIb fibers has been confirmed by RT-PCR analyses on single
muscle fibers (8). It is thus likely that the low-mobility MHC band in
human muscle in fact corresponds to a type IIx MHC species rather than
to a type IIb MHC species. The existence of a type IIb MHC in human
skeletal muscle remains to be established.
The other important finding in this study was that the proportion of
type I (/slow) myosin increased in patients with severe COPD at the
expense of type II (fast) isoforms. Although the role of decreased
thyroid hormone plasma concentrations in this increased proportion of
slow myosin cannot be totally ruled out, it seems most unlikely in
stable well-nourished COPD patients in whom such a decrease has not
been reported to date. Furthermore, the correlations we found between
the proportion of slow myosin and the parameters of respiratory
function do not support this hypothesis. Indeed, to get insights into
the mechanisms responsible for the increased proportion of slow myosin,
we looked for correlations between this proportion and the parameters
of respiratory function. In this respect, it should be noted that our
aim was not to assess diaphragm muscle contractile properties but
rather to measure the parameters of respiratory function that may
testify for the severity and duration of the increase in respiratory
load and lung volume. By doing so, our goal was just to bring together, in patients with various degrees of airway obstruction and lung distension, a biochemical parameter involved in muscle adaptation to
load and the parameters testifying for chronic increase in muscle load.
Our hypothesis was that the latter alterations, which have been shown
to chronically increase the load imposed on the diaphragm, will result
in the need for a change in myosin phenotype as observed in the heart
submitted to chronic hemodynamic overload (9, 17, 18). Although control
and COPD patients alone did not allow us to observe significant
correlations, the pooling of the two subject categories, by including a
whole range of values of slow myosin percentages and functional
parameters, allowed us to observe positive correlations between the
proportion of slow myosin and both TLC and FRC and a negative
correlation with FEV1. Although no
absolute cause-to-effect relationship can be ascertained from our
studies, these correlations suggested that the degree of the
fast-to-slow myosin transition increases with lung distension and
airway obstruction. In this respect, it is interesting to note that the
strongest correlation was observed with FRC, which determines the
length of the diaphragm before inspiration. As FRC increases, the
diaphragm shortens, which diminishes its capacity to generate the
driving pressure of the respiratory system (29).
In the present study, the fact that myosin subtype correlated with the reported functional parameters does not mean that the MHC phenotype determines these physiological parameters but rather that the higher the airway obstruction and lung distension, the higher the proportion of slow myosin. This myosin phenotype conversion is reminiscent of the changes in myosin phenotype in fast skeletal muscle facing unusual functional requirements (30) and those occurring in the ventricles of small rodents (18) and in human atria (9, 17) subjected to a chronic increase in the hemodynamic load. However, the precise nature of the triggering factor(s) involved in myosin phenotypic alteration in the flattened and/or overloaded human diaphragm remains to be elucidated. The shortening of diaphragm operating length, which results from its flattening in patients with COPD, and the resulting detrimental mechanical and energetical conditions under which the muscle has to cope with an increased respiratory load could be one of the triggers. However, we cannot exclude that other triggers are also responsible. For instance, changes in the frequency of stimulation are also an important trigger for myosin phenotypic alteration in skeletal muscle (30). Culture of cardiac myocytes and isolated perfused hearts has been very fruitful in determining a number of triggers and neurohumoral factors involved in myocyte phenotypic changes during mechanical overload of the heart, pointing to the important role of myocyte stretch acting or not acting through an autocrine production of angiotensin II, depending on the developmental stage considered (11, 31). The use of differentiated skeletal muscle in tissue culture has also allowed the demonstration of the role of mechanical stimulation and the involvement of various growth factors and signal transduction pathways in skeletal muscle remodeling (20, 33). Further studies will be needed to determine which among these various mechanisms and pathways are operating in the chronically overloaded human diaphragm.
In conclusion, this study extends the concept of myosin plasticity to the diaphragm, the main muscle involved in the control of ventilatory function. Like other striated muscles, gene expression in the diaphragm can thus adapt to new functional requirements. Other possible changes such as hypertrophy of a specific fiber type (type I?) or modifications of diaphragm blood supply or capillary bed density in the overloaded diaphragm remain to be identified. However, the present data provide new information concerning the relationship between the functional properties of the human diaphragm and its biochemical and molecular composition. The changes observed at the molecular level in the human diaphragm of COPD patients are in-line with a previous physiological study (27) that found that diaphragmatic force is well preserved in severe COPD in a stable state. Taken together, our data and the physiological data previously described (27) raise the question of the usefulness of the therapeutic approach based on respiratory muscle rehabilitation currently used in COPD patients.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank David Young for help in restyling the manuscript.
![]() |
FOOTNOTES |
---|
This work was supported in part by grants from the Association Française contre les Myopathies and Agenzia Spaziale Italania.
Address for reprint requests: M. Aubier, Unité de Pneumologie, Hôpital Bichat, 46, rue Henri Huchard, 75018 Paris, France.
Received 26 February 1997; accepted in final form 5 January 1998.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Alpert, N. R.,
and
L. A. Mulieri.
Increased myothermal economy of isometric force generation in compensated cardiac hypertrophy induced by pulmonary artery constriction in the rabbit.
Circ. Res.
50:
491-500,
1982[Medline].
2.
Bär, A.,
and
D. Pette.
Three fast myosin heavy chains in adult rat skeletal muscle.
FEBS Lett.
235:
153-155,
1988[Medline].
3.
Barany, M.
ATPase activity of myosin correlated with speed of muscle contraction.
J. Gen. Physiol.
50:
197-216,
1967
4.
Biral, D.,
R. Betto,
D. Danieli-Betto,
and
G. Salviati.
Myosin heavy chain composition of single fibers from normal human muscle.
Biochem. J.
250:
307-308,
1988[Medline].
5.
Danieli-Betto, D.,
E. Zerbato,
and
R. Betto.
Type 1, 2A and 2B myosin heavy chain electrophoretic analysis of rat muscle fibers.
Biochem. Biophys. Res. Commun.
138:
981-987,
1986[Medline].
6.
Delcayre, C.,
and
B. Swynghedauw.
A comparative study of heart myosin ATPase and light subunits from different species.
Pflügers Arch.
355:
39-47,
1975[Medline].
7.
DeNardi, C.,
S. Ausoni,
P. Moretti,
L. Gorza,
M. Velleca,
M. Buckingham,
and
S. Schiaffino.
Type 2X myosin heavy chain is coded by a muscle fiber type-specific and developmentally regulated gene.
J. Cell Biol.
123:
823-835,
1993[Abstract].
8.
Ennion, S.,
J. Sant'Ana Pereira,
A. J. Sargeant,
A. Young,
and
G. Goldspink.
Characterization of human skeletal muscle fibers according to the myosin heavy chains they express.
J. Muscle Res. Cell Motil.
16:
35-43,
1995[Medline].
9.
Gorza, L.,
J. J. Mercadier,
K. Schwartz,
L. E. Thornell,
S. Sartore,
and
S. Schiaffino.
Myosin types in the human heart. An immunofluorescence study of normal and hypertrophied atrial and ventricular myocardium.
Circ. Res.
54:
694-702,
1984[Abstract].
10.
Hoh, J. F. Y.,
P. A. McGrath,
and
P. T. Hale.
Electrophoretic analysis of multiple forms of rat cardiac myosin: effect of hypophysectomy and thyroxine replacement.
J. Mol. Cell. Cardiol.
10:
1053-1076,
1978[Medline].
11.
Kent, R. L.,
and
P. J. McDermott.
Passive load and angiotensin II evoke differential responses of gene expression and protein synthesis in cardiac myocytes.
Circ. Res.
78:
829-838,
1996
12.
Klitgaard, H.,
M. Zhou,
S. Schiaffino,
R. Betto,
G. Salviati,
and
B. Saltin.
Ageing alters the myosin heavy chain composition of single fibers from human skeletal muscles.
Acta Physiol. Scand.
140:
55-62,
1990[Medline].
13.
LaFramboise, W. A.,
J. F. Watchko,
B. S. Brozanski,
M. J. Daood,
and
R. D. Guthrie.
Myosin heavy chain expression in respiratory muscles of the rat.
Am. J. Respir. Cell Mol. Biol.
6:
335-339,
1992[Medline].
14.
LaFramboise, W. A.,
M. J. Daood,
R. D. Guthrie,
G. S. Butler-Browne,
R. G. Whalen,
and
M. Ontell.
Myosin isoforms in neonatal rat extensor digitorum longus, diaphragm, and soleus muscles.
Am. J. Physiol.
259 (Lung Cell. Mol. Physiol. 3):
L116-L122,
1990
15.
Lompré, A. M.,
J. J. Mercadier,
C. Wisnewsky,
P. Bouveret,
C. Pantaloni,
A. d'Albis,
and
K. Schwartz.
Species and age-dependent changes in the relative amounts of cardiac myosin isoenzymes in mammals.
Dev. Biol.
84:
286-290,
1981.
16.
Macklem, P. T.
Respiratory muscles: the vital pump.
Chest
78:
753-758,
1980[Medline].
17.
Mercadier, J. J.,
D. de la Bastie,
P. Ménashé,
A. N'Guyen Van Cao,
P. Bouveret,
P. Lorente,
A. Piwnica,
R. Slama,
and
K. Schwartz.
Alpha-myosin heavy chain isoform and atrial size in patients with various types of mitral valve dysfunction: a quantitative study.
J. Am. Coll. Cardiol.
9:
1024-1030,
1987[Medline].
18.
Mercadier, J. J.,
A. M. Lompré,
C. Wisnewsky,
J. L. Samuel,
J. Bercovici,
B. Swynghedauw,
and
K. Schwartz.
Myosin isoenzymic changes in several models of rat cardiac hypertrophy.
Circ. Res.
49:
525-532,
1981[Abstract].
19.
Pardy, R. L.,
and
C. Roussos.
Endurance of hyperventilation in chronic airflow limitation.
Chest
83:
744-750,
1983[Abstract].
20.
Perrone, C. E.,
D. Fenwick-Smith,
and
H. H. Vandenburgh.
Collagen and stretch modulate autocrine secretion of insulin-like growth factor-1 and insulin-like growth factor binding proteins from differentiated skeletal muscle cells.
J. Biol. Chem.
270:
2099-2106,
1995
21.
Roy, R. K.,
I. D. Meadows,
K. M. Baldwin,
and
V. R. Edgerton.
Functional significance of compensatory overloaded rat fast muscle.
J. Appl. Physiol.
52:
473-478,
1982
22.
Saggin, L.,
S. Ausoni,
L. Gorza,
S. Sartore,
and
S. Schiaffino.
Troponin T switching in the developing rat heart.
J. Biol. Chem.
263:
18488-18492,
1988
23.
Schiaffino, S.,
L. Gorza,
L. Saggin,
C. Valfré,
and
S. Sartore.
Myosin changes in hypertrophied human atrial and ventricular myocardium. A correlated immunofluorescence and quantitative immunochemical study on serial cryosections.
Eur. Heart J.
75:
95-102,
1984.
24.
Schiaffino, S.,
L. Gorza,
S. Sartore,
L. Saggin,
M. Vianello,
K. Gundersen,
and
T. Lomo.
Three myosin heavy chain isoforms in type 2 skeletal muscle fibers.
J. Muscle Res. Cell Motil.
10:
197-205,
1989[Medline].
25.
Schiaffino, S.,
and
C. Reggiani.
Myosin isoforms in mammalian skeletal muscle.
J. Appl. Physiol.
77:
493-501,
1994
26.
Schwartz, K.,
Y. Lecarpentier,
J. L. Martin,
A. M. Lompré,
J. J. Mercadier,
and
B. Swynghedauw.
Myosin isoenzymic distribution correlates with speed of myocardial contraction.
J. Mol. Cell. Cardiol.
13:
1071-1075,
1981[Medline].
27.
Similowski, T.,
S. Yan,
P. Gauthier,
P. T. Macklem,
and
F. Bellemare.
Contractile properties of the human diaphragm during chronic hyperinflation.
N. Engl. J. Med.
325:
917-923,
1991[Abstract].
28.
Smerdu, V.,
I. Karsch-Mizrachi,
M. Campione,
L. Leinwand,
and
S. Schiaffino.
Type IIx myosin heavy chain transcripts are expressed in type IIb fibers of human skeletal muscle.
Am. J. Physiol.
267 (Cell Physiol. 36):
C1723-C1728,
1994
29.
Smith, J.,
and
F. Bellemare.
Effect of lung volume on in vivo contraction characteristics of human diaphragm.
J. Appl. Physiol.
62:
1893-1900,
1987
30.
Swynghedauw, B.
Developmental and functional adaptation of contractile proteins in cardiac and skeletal muscles.
Physiol. Rev.
66:
710-771,
1986
31.
Thienelt, C. D.,
E. O. Weinberg,
J. Bartunek,
and
B. H. Lorell.
Load-induced growth responses in isolated adult rat hearts. Role of the AT1 receptor.
Circulation
95:
2677-2683,
1997
32.
Towbin, H.,
T. Staehelin,
and
J. Gordon.
Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.
Proc. Natl. Acad. Sci. USA
16:
4350-4354,
1979.
33.
Vandenburgh, H. H.,
J. Shansky,
R. Solerssi,
and
J. Chromiak.
Mechanical stimulation of skeletal muscle increases prostaglandin F2 alpha production, cyclooxygenase activity, and cell growth by a pertussis toxin sensitive mechanism.
J. Cell. Physiol.
163:
285-294,
1995[Medline].
34.
Whalen, R. G.,
S. M. Sell,
G. S. Butler-Browne,
K. Schwartz,
P. Bouveret,
and
I. Pinset-Härström.
Three myosin heavy-chain isozymes appear sequentially in rat muscle development.
Nature
292:
805-809,
1981[Medline].