Vol. 92, Issue 3, 1074-1082, March 2002
Effects of postnatal maturation on energetics and
cross-bridge properties in rat diaphragm
Gilles
Orliaguet1,
Olivier
Langeron2,
Belaid
Bouhemad2,
Pierre
Coriat2,
Yves
LeCarpentier3, and
Bruno
Riou2,4
1 Department of Anesthesiology and Critical Care, Centre
Hospitalo-Universitaire Necker-Enfants Malades, Assistance
Publique-Hôpitaux de Paris, Université Paris V 75743 Paris
Cedex 15; 2 Department of Anesthesiology and Critical Care,
4 Department of Emergency Medicine and Surgery, Centre
Hospitalo-Universitaire Pitié-Salpêtrière, Assistance
Publique-Hôpitaux de Paris, Université Pierre et Marie
Curie 75651 Paris Cedex 13; 3 Department of Physiology, Centre
Hospitalo-Universitaire Bicêtre, Assistance
Publique-Hôpitaux de Paris, Université Paris XI, Le
Kremlin-Bicêtre 94270, France
 |
ABSTRACT |
The effects of maturation on
cross-bridge (CB) properties were studied in rat diaphragm strips
obtained at postnatal days 3, 10, and
17 and in adults (10-12 wk old). Calculations of muscle energetics and characteristics of CBs were determined from standard Huxley equations. Maturation did not change the curvature of the force-velocity relationship or the peak of mechanical efficiency. There
was a significant increase in the total number of CBs per cross-sectional area (m) with aging but not in single CB force. The
turnover rate of myosin ATPase increased, the duration of the CB cycle
decreased, and the velocity of CBs decreased significantly only after
the first week postpartum. There was a linear relationship between
maximum total force and m (r = 0.969, P < 0.001), and between maximum unloaded shortening velocity and m
(r = 0.728, P < 0.001). When this
study in the rat and previous study in the hamster are compared, it
appears that there are few species differences in the postnatal
maturation process of the diaphragm.
skeletal muscle; development; cross-bridge cycling
| |
INTRODUCTION |
THE DIAPHRAGM MUSCLE
is the principal inspiratory muscle, chronically active from birth and
generating adequate force to sustain ventilation. Thus it is the only
skeletal muscle that may be strictly regarded as being essential.
During postnatal maturation, in diaphragm as well as in other skeletal
muscles, major ultrastructural, biochemical, and metabolic changes
occur, resulting in an improved contractility (9, 20, 30, 31, 38,
41, 46). However, the precise mechanisms by which maturation
induces changes in the contractile performance of diaphragm muscle
remain incompletely understood (8, 12, 19, 31, 47).
In diaphragm muscle, as well in other striated muscles,
mechanical processes result from the cyclic interaction between two contractile proteins, actin and myosin. The mechanical and energetic properties of the muscle depend on actomyosin cross-bridge (CB) cycling
because CBs produce a power stroke that drives the myosin molecules
along the actin filaments (17, 18).
According to the most widely accepted theory of muscle contraction
(17, 18), CBs act as independent force generators, and
muscle force depends on both the elementary force produced per single
CB and the total number of CBs formed (17, 18). In a
recent study performed in the hamster, Coirault et al. (8) have suggested that developmental changes in diaphragm muscle force
were associated with changes in CB number and kinetics but not with
changes in the elementary force produced per single CB or in mechanical
efficiency. However, species differences regarding changes in CB
properties during postnatal maturation might exist, although neonatal
rats have become widely used as experimental laboratory animals,
especially in cardiorespiratory physiological and pharmacological
fields (3, 37, 38, 43, 46). Moreover, Coirault et al.
(8) have studied only 1-day- and 1-wk-old hamsters, whereas it may be important to study additional intermediate stages during postnatal maturation. This study may be especially relevant in
the rat, whose transition from a fetal to an adult pattern of
contractile protein isoforms has been reported to be more complex and
delayed compared with other mammal species (22, 27). In fact, at term, the rat diaphragm muscle myosin heavy chain (MHC) phenotype is a composite of neonatal MHC (MHCneo; 66%),
MHCslow (12%) and MHC2A (22%). In contrast,
at birth, the human diaphragm is more mature, being composed of more
fast adult MHC than the rat diaphragm, with 15% MHCneo,
32% MHCslow, 47% MHC2A, and 6% MHC2B (27).
We, therefore, conducted an in vitro study on the effects of
postnatal maturation on diaphragm muscle CB properties of 3-day-old, 10-day-old, 17-day-old, and adults rats. We hypothesized that, despite
marked changes in the contractile protein isoform composition of the
rat diaphragm muscle during postnatal development, there are no changes
in unitary force production per CB but that the increase in the total
number of CBs per cross-sectional area with aging contributes to the
developmental changes in contractility.
| |
METHODS |
Animals and study design.
Care of the animals conformed to the recommendations of the Helsinski
Declaration, and the study was performed in accordance with the
regulations laid down by the French Ministry of Agriculture. After
birth, rat pups were kept in cages with their mothers. Adult rats
received rat chow and water ad libitum. A 12-h light-dark cycle was
provided. Experiments were performed on Wistar rats aged 3 days
(n = 20), 10 days (n = 20), 17 days
(n = 20), and 10-12 wk (adult, n = 20).
After a brief anesthesia with ether, a median laparotomy was
performed and a muscle strip from the ventral costal diaphragm was
carefully dissected from the muscle in situ, as previously reported
(7). With this procedure, diaphragmatic fibers were parallel and of approximately equal length (7). This
diaphragm strip was vertically suspended in a 200-ml jacketed reservoir with Krebs-Henseleit bicarbonate buffer solution that contained (in mM)
118 sodium chloride, 4.7 potassium chloride, 1.2 magnesium sulfate, 1.1 dipotassium hydrogen phosphate, 25 sodium hydrogen carbonate, 2.5 calcium chloride, and 4.5 glucose. The jacketed reservoir was
maintained at 29°C with continuous monitoring of the solution
temperature. The bathing solution was bubbled with 95% oxygen-5%
carbon dioxide, resulting in a pH of 7.40. Preparations were field
stimulated with 1-ms rectangular pulses, at a rate of 50 Hz for 300 ms,
to induce a tetanic contraction (10 contractions/min). A frequency of
stimulation of 50 Hz was used because it assures a maximal tetanus
across all age groups without increasing the risk of inducing
high-frequency fatigue (29). After a 30-min stabilization
period, at the initial muscle length at the apex of the length-active
isometric tension curve (Lmax), diaphragm muscle
strips recovered their optimal mechanical performance (7). At the end of the study, the cross-sectional area (in mm2)
was calculated from the ratio of fresh muscle weight to muscle length
at Lmax, assuming a muscle density of
1.06. Body weight was measured at the moment the animal was killed.
Electromagnetic lever system.
The electromagnetic lever system has been previously described
(7, 23, 33). Briefly, the load applied to the muscle was
determined by means of a servomechanism-controlled current through the
coil of an electromagnet. Muscular shortening induced a displacement of
the lever, which modulated the light intensity of a photoelectric
transducer. The initial preload (resting force), which determined
Lmax, was automatically maintained constant
throughout the experiment. All analyses were made from digital records
of force and length obtained with a computer, as previously described (7, 23).
Mechanical parameters.
The main mechanical parameters were calculated from three
consecutive tetanic contractions preloaded at
Lmax. The first contraction was isotonic and
loaded with preload only. The second contraction was loaded with
preload and was abruptly clamped to zero load immediately after the
electrical stimulus, according to the zero-load clamp technique
(4). The third contraction was fully isometric at
Lmax. The maximum extent of shortening
(
L) and maximum lengthening velocity (Vr) were
determined from the first contraction. The maximum unloaded shortening
velocity (Vmax), as calculated by means of the
zero-load clamp technique, was determined from the second contraction.
The maximum active isometric force normalized per cross-sectional area
(AF), and the peak of the positive (+) and negative (
) maximum
contraction rate (dF/dt) force derivatives normalized per
cross-sectional area were determined from the third contraction (Fig.
1).
View larger version (20K):
[in this window]
[in a new window]
|
Fig. 1.
Mechanical parameters of contraction and relaxation.
Top: muscle shortening length
(L/Lmax) plotted vs. time.
Bottom: force (mN/mm2) plotted vs. time. Tetanus
1 was isotonic and loaded with preload only to determine the maximum
extent of shortening (L) and maximum lengthening velocity
(Vr). Tetanus 2 was loaded with preload and was abruptly
clamped to zero load immediately after the electrical stimulus to
determine the maximum unloaded shortening velocity
(Vmax). Tetanus 3 was fully isometric at the
longest length measured (Lmax) to determine the
maximum active force (AF), and the peak of the positive
(+dF/dt) and negative (dF/dt) force
derivatives.
|
|
Vmax, AF, L, and
+dF/dt tested the contraction phase (inotropy).
Vr and dF/dt tested the relaxation phase.
Nevertheless, because changes in the contraction phase induce
coordinated changes in the relaxation phase, relaxation parameters
cannot assess lusitropy, and, therefore, variations in contraction and
relaxation must be considered simultaneously (7, 23). Thus
we calculated the ratios Vr/L and
(dF/dt)/AF, which assessed lusitropy under isotonic and
isometric conditions, respectively.
Energetic parameters and CB properties.
Calculations of muscle energetics and characteristics of CBs were
determined from Huxley's equations (17), as previously described (5, 8, 24-26). The force-velocity
(F-V) relationship was derived from the peak velocity
(V) of various afterloaded contractions, plotted against the
isotonic load level normalized per cross-sectional area (F) from zero
load to isometric force (Fig. 2).
Experimental data of the F-V curve were fitted according to
Hill's equation (15)
|
(1)
|
where cFmax is the calculated peak isometric force for
V = 0, and a and b are the asymptotes of the
hyperbola. The curvature (G) of the F-V relationship was
|
(2)
|
where cVmax is the calculated peak
V at zero load.
View larger version (31K):
[in this window]
[in a new window]
|
Fig. 2.
A typical 10-day-old diaphragm muscle contracting at 10 different afterload levels from zero load to isometric load and
stimulated in tetanus (50 Hz, train duration = 350 ms).
Lmax is the initial muscle length corresponding
to the apex of the length-force curve. Top:
L/Lmax plotted vs. time.
Bottom: force (mN/mm2) plotted vs. time.
|
|
The Huxley equations were used to calculate the rate of total energy
release (E), the isotonic force (PHux), and the rate of
mechanical work (WM) as a function of V, as
previously reported (5, 8, 24-26). E is given as
|
(3)
|
where m is the number of CB per square millimeter at maximum
PHux, f1 is the maximum value of the rate
constant for CB attachment, and g1 and g2 are
the peak values of the rate constants for CB detachment. The
instantaneous movement (x) of the myosin head relative to actin varies
from h to 0. The step size of the CB (h) is defined by the
translocation distance of the actin filament per ATP hydrolysis and
produced by the swing of the myosin head; f1 and
g1 correspond to x = h, and g2 corresponds
to x < 0; e is the free energy required to split one ATP
molecule, l is the distance between two actin sites, and
= (f1 + g1) × h/2 = b. Calculations of f1, g1, and g2 are
given by the following equations
![f<SUB>1</SUB><IT>=</IT>[(g<SUB>1</SUB><SUP>2</SUP><IT>+</IT>4g<SUB>1</SUB><IT>·</IT>g<SUB>2</SUB>)<SUP>½</SUP><IT>−</IT>g<SUB>1</SUB>]<IT>/</IT>2](/content/vol92/issue3/fulltext/1074/img005.gif)
|
(4)
|
|
(5)
|
|
(6)
|
The maximum value of total energy release occurs at
Vmax. The minimum value of the rate of total
energy release (E0) occurs under isometric conditions and
is equal to the product a × b and is also given by the following
equation
|
(7)
|
The maximum turnover rate of myosin ATPase in isometric
conditions (kcat, s
1) is given by
the following equation
|
(8)
|
Assuming that one molecule of ATP is split in each CB cycle, the
total duration of the time cycle (tc), the total
CB cycle duration (tc = 1/kcat), the duration of the power stroke [time stroke (ts)], the duty ratio
(ts/tc), and the mean
velocity of each CB (v0) were calculated as
|
(9)
|
|
(10)
|
PHux is given by
|
(11)
|
where w is the WM of a unitary CB. The elementary
force per unitary CB in isometric conditions (
, pN) is given by the
following equation
|
(12)
|
WM is given by
|
(13)
|
At any given load, the mechanical efficiency (Eff) of the muscle
is defined as the ratio of WM to E, and Effmax
is the maximum value of Eff.
A stroke size (h) of 11 nm has been determined by means of optical
tweezers (11) and is supported by the three-dimensional structure of crystallized myosin head (10, 35). The
distance l is equal to 36 nm (36). The free
energy required to split one ATP molecule is 5.1 × 1020 J. Because w is 0.75e, the value of w is 3.8 × 1020 J (48).
Statistical analysis.
Data are expressed as means ± SD. Comparisons of several means
were performed by using one-way ANOVA and Newman-Keuls test. F-V relationship was fitted to a hyperbola by using
multilinear regression and the least-squares method. Correlation
between two variables was performed by using the least-squares method.
All P values were two-tailed, and a P value of
<0.05 was required to reject the null hypothesis. Statistical analysis
was performed with the use of NCSS 6.0 software (Statistical Solutions,
Cork, Ireland).
| |
RESULTS |
As shown in Table 1, we observed
significant differences in body weight, diaphragm strip weight,
section, and Lmax.
Main mechanical parameters.
During postnatal maturation, we observed significant increases in
mechanical parameters testing inotropy in isotonic
(Vmax) and isometric (AF, +dF/dt)
conditions (Table 2). Vr and
dF/dt, which tested relaxation in isotonic (Vr)
and isometric conditions (dF/dt) were also significantly
modified during postnatal maturation (Table 2). The ratio
Vr/L was only significantly different between 3-day-old
and adult rats (Table 2). The ratio (dF/dt)/AF, was significantly lower in 3- and 10-day-old rats than in other groups (Table 2).
Energetic characteristics.
Postnatal maturation did not significantly change the curvature G of
the F-V relationship (Table
3). There was a significant increase in
the asymptote a and b of the hyperbola with postnatal maturation
(Table 3). The peak of mechanical efficiency (Effmax) was
not modified by postnatal maturation, although there was a significant
increase in the rate of total energy release (Table 3).
CB properties.
During postnatal maturation, there was a significant increase in the
total number of CBs per cross-sectional area but not in the force
developed by unitary CB (Table 4). The
kcat was only significantly modified by
maturation in adult rats (Table 4). Moreover, the maximum values of the
rate constant for CB attachment and detachment significantly increased
during postnatal maturation (Table 4). The maximum value of the rate
constant for CB detachment was only significantly modified by
maturation in adult rats (Table 4). Postnatal maturation was associated with a decrease in the total duration of the CB cycle and
ts, associated with a decrease in the duty ratio
(Table 4). An increase in the v0 of CBs was also
observed during maturation (Table 4).
Relationships between the mechanical and energetic parameters.
There was a strong linear relationship (r = 0.969, P < 0.001) between maximum total isometric force and m
(Fig. 3), with the number of CBs
increasing proportionally with force. There was also a significant
correlation (r = 0.728, P < 0.001)
between Vmax and m (Fig. 3). Conversely, there
was neither significant correlation between AF and [r = 0.252, P value not significant (NS)]
nor between Vmax and (r = 0.161, NS). A weak correlation (r = 0.541, P < 0.001) was noted between
Vmax and kcat (Fig.
4). With the use of multiple correlation,
it appeared that Vmax was more strongly
correlated with age (r = 0.780, P < 0.001) than with kcat (r = 0.541, P = 0.006).
View larger version (18K):
[in this window]
[in a new window]
|
Fig. 3.
Significant correlations between maximum isometric total
force (TF) and the total number of cross bridges (m) (A),
and Vmax and m (B).
|
|
View larger version (23K):
[in this window]
[in a new window]
|
Fig. 4.
Significant but weak correlation between
Vmax and turnover rate of myosin ATPase per
myosin site (kcat).
|
|
| |
DISCUSSION |
In our study, CB properties were calculated from mechanical data
obtained in isolated diaphragm strips by using Huxley's equations (17). Huxley's theory (17) remains the most
commonly accepted theory of muscle contraction, and his equations have
been applied in studies on the effects of different pathophysiological
conditions (congestive heart failure, fatigue) or treatment
administration (angiotensin-converting enzyme inhibition, nandrolone)
on diaphragm muscle CB kinetics of various animal species (mouse,
rabbit, rat) (5, 24-26, 40). The effects of postnatal
maturation on physical characteristics and mechanical parameters
of contraction, relaxation, and contraction-relaxation coupling of
rat diaphragm observed in our study (Tables 1 and 2) are in agreement
with those previously described in other rat diaphragm studies
(12, 19, 46, 49).
Shortening velocity.
Vmax increased significantly during postnatal
maturation (Table 2), as previously reported (19). Some
authors have proposed that these changes could be largely related to
postnatal transitions in MHC isoform expression, especially the
progressive decrease in MHCneo isoform expression and the
progressive increase in MHC2X and MHC2B
expression (19). In fact, MHC isoforms differ in their ATPase activity (47), and it has been suggested that these
differences in actomyosin ATPase activity contribute to the
relationship between MHC phenotype transitions and velocities of the
diaphragm during early postnatal development (1, 19, 47).
A lower actomyosin ATPase activity has been reported in newborn rats,
compared with adult rats, with actomyosin ATPase activity depending on
MHC isoform expression (47). The lowest actomyosin ATPase
activity was observed in those fibers that expressed MHCneo
and MHCslow, and it increased in rank order in IIa > IIx < IIb fibers (47). In contrast, other studies
have suggested that there is only a weak correlation between MHC
isoform expression and changes in diaphragmatic velocity during maturation, supporting the hypothesis that factors in addition to the
postnatal transitions in MHC isoform expression are involved in
regulating a diaphragmatic increase in velocity of shortening (34).
In the hamster diaphragm, Coirault et al. (8) did not
observe any significant correlation between kcat
and Vmax during postnatal maturation. In
contrast, in rat diaphragm, we observed a weak but significant
correlation between kcat and
Vmax. It should be pointed out that the number
of animals studied (n = 80) is likely to have provided
us a sufficient statistical power to detect this weak correlation
(8). Our results suggest that the mechanisms underlying
postnatal changes in Vmax are only partly
dependent on actomyosin ATPase activity (1). In addition,
according to Huxley's theory, kcat is
principally governed by f1 and g1, whereas Vmax is proportional to g2.
Therefore, complex changes in the rate constants of CB attachment and
detachment during postnatal maturation (Table 4) may partly explain the
weak correlation between Vmax and
kcat. We have observed that
kcat did not vary significantly during the first
2 wk postpartum, whereas Vmax increased significantly from day 3 to day 17 and adulthood
(Table 4). Accordingly, changes in the tc,
assuming that one ATP molecule is hydrolyzed per CB cycle, were
inversely related to developmental changes in
kcat. These results agree with those by Coirault
et al. (8) and, as suggested by these authors, may be
related to the progressive disappearance of MHCneo isoforms
and the expression of adult fast myosin isoforms. This hypothesis is
consistent with biochemical studies (2, 16) and indicates
that the overall cycle of ATP splitting takes place more slowly in
immature than in adult diaphragms. Indeed, tc
decreased significantly only in adult rats (Table 4). During the first
2 wk postpartum, there is an important increase in MHCslow
and a concomitant reduction in the proportion of MHCneo (5, 9, 19, 47, 49). However, both MHCneo and
MHCslow have a low actomyosin ATPase activity
(47), suggesting that the increase in MHCslow
counterbalances the decrease in MHCneo under circumstances
in which myosin ATPase activity is concerned, and explains why
kcat values remained unchanged between
days 3 and 17 postpartum but increased
thereafter. This is consistent with the results obtained for both
f1 and g1 (Table 4). According to Huxley's
equations, kcat is principally governed by the
two rate constants, f1 and g1
(17).
Active force and .
The values calculated in our study (Table 4) agree with those
previously calculated from Huxley's equations for adult hamster (8, 25) and rat diaphragm (26). In addition,
values calculated in our study are also in the range of the average
force between an actin filament and a single molecule of myosin as
measured by pulling the filament with optical tweezers
(32). During postnatal maturation, we observed a
significant increase in diaphragmatic force and in the total number of
CBs, as well as a strong linear relationship between maximum total
isometric force and the total number of CBs (Fig. 3), whereas no change
in was observed. Because the maximum total isometric force is the
product of the number of CBs and (17), these results
suggest that the increase in force of the developing diaphragm muscle
are mainly related to the increase in the number of CBs. This
hypothesis is in agreement with the results of Coirault et al.
(8), obtained in hamster diaphragm. The total number of
CBs per square millimeter reflects the cross-sectional density of CBs
of a given muscle. During diaphragm postnatal maturation, different
mechanisms may participate in the increase in the total number of CBs,
in parallel with an increase in myofibrillar protein density
(30), a reduction in the percentage of interstitial space
relative to total muscle cross-sectional area (14), and an
increase in fiber cross-sectional area (41). The lack of
change in force per single CB observed in our study suggests that
developmental increase in maximum total isometric force is not related
to reorientation of oblique fibers into the longitudinal axis of the
diaphragm muscle, as previously suggested by Coirault et al.
(8). It should be pointed out that our methodology enables
us only to calculate the number of active CBs but not the total number
of CBs present. Indeed, we cannot make the difference between a
decreased total number of CBs, leading to a decreased force, and a
decrease in calcium available for contraction, also leading to a
decreased force through a decrease in the number of active CBs. Because
diaphragmatic contraction highly depends on intracellular calcium,
mainly from the sarcoplasmic reticulum, rather than extracellular
calcium, the maturation of the sarcoplasmic reticulum may have played
an important role in the postnatal increase in force and number of
active CBs observed in our study. Ryanodine receptors (RyR) are
intracellular homotetrameric Ca2+-release channels, whose
subunits are encoded by three different genes indicated as RyR1, RyR2,
and RyR3. In adult skeletal and diaphragmatic muscles, RyR1 is
essential in triggering contraction. Expression of RyR1 requires about
3-4 wk to reach the high levels that are maintained throughout
adult life (21). Another isoform, RyR3, more expressed in
the diaphragm than in other skeletal muscles (44), is
predominantly expressed during fetal and neonatal development and has
been shown to play a physiological role in excitation-contraction coupling of neonatal skeletal muscles (3, 44). RyR3 is
already expressed during fetal development, but its expression is
maximum during the neonatal phase (2-15 days) in the rat
(44). Therefore, the changes in CB cycling observed
following the 2 wk postpartum may be in relation with the progressive
disappearance of RyR3. Alternatively, the changes in force generation
during postnatal maturation may be related to the MHC content per half
sarcomere (13). In a recent study, maximum force values of
rat diaphragm muscle bundles and single fibers were normalized for MHC
content per half sarcomere to determine the effect of CB number on
maximum specific force during maturation (13). MHC content
per half sarcomere progressively increased during early postnatal
maturation, but no change in force per half sarcomere MHC content was
noted between days 0 and 14, excepted for fibers
predominantly expressing MHC2X, which represent about 12%
of total MHC content at day 14 and 0% between days
0 and 7 (47, 49). These results indicate that the difference in specific force mainly reflects differences in
MHC content per half sarcomere. Our results are in agreement with this
assumption because we observed that, during postnatal development,
there was no increase in the unitary force per CB but that there was an
increase in the total number of CB, which may reflect the increase in
MHC content per half sarcomere.
Mechanical efficiency.
We observed no significant changes in peak mechanical efficiency during
postnatal maturation (Table 3). However, changes in myofibrillar ATPase
activity are usually thought to be responsible for changes in the
economy of the muscle force generation and/or in peak mechanical
efficiency (48). It has been shown that slow-contracting muscles such as soleus, which has a low ATPase activity, have a greater
economy of force generation than fast-contracting muscles with high
ATPase activity (48). Accordingly, higher ATPase activity in adult diaphragm would be expected to decrease both maximum efficiency and the G curvature of the F-V relationship. In
contrast, we observed no such changes during maturation in the rat
(Table 3), as previously reported in hamster diaphragm muscle
(8), despite a significant increase in
kcat. These results indicate that changes in
myosin ATPase activitity are not always associated to changes in
contractile efficiency, as previously observed in cardiac muscle
(42).
Relaxation.
We also observed a progressive change in diaphragm relaxation during
postnatal maturation (Table 2). However, these changes were mainly
significant after the second week postpartum, as previously reported
(45). The precise mechanisms by which postnatal maturation modified diaphragm relaxation remain unclear. Relaxation is controlled by a complex interplay between inactivation and loading conditions. The
rate of inactivation is limited mainly by active Ca2+
pumping by the sarcoplasmic reticulum, Ca2+ removal from
troponin C, and the instantaneous number of working CBs
(6). Thus the increase in the total number of CBs noted in
our study (Table 4) may have played a role in the improvement in
relaxation observed during postnatal maturation by increasing the
instantaneous number of working CBs. In addition, there is some
evidence suggesting that active calcium movements by the sarcoplasmic
reticulum could be altered in the neonatal diaphragm. Maxwell et al.
(30) showed evidence of an immature sarcoplasmic reticulum
in fibers from premature baboon diaphragms. These factors could limit
capacities for Ca2+ release and reuptake in the diaphragm
muscle. On the other hand, it has been suggested that CB kinetics have
a limited influence on the overall time course of diaphragm relaxation
(6).
CB kinetics.
We observed marked differences in CB kinetics between the adult rat and
the adult hamster (8). Although the value of a single CB
was similar in the two species, in agreement with Huxley's theory, the
number of CBs, and the constants of attachment (f1) and
detachment (g1, g2) were lower in the rat than
in the hamster (8). Despite these marked differences, we
observed no important species difference in the postnatal maturation
process concerning CB kinetics. This result suggests that postnatal
maturation involves common mechanisms that do not markedly differ from
one species to another. Nevertheless, further investigations on other
mammal species should be performed to confirm this hypothesis.
Limitations of the study.
The design and methodology of our study do not allow us to analyze CB
kinetics at the single-fiber level or in relation to the differences in
MHC expression. In muscle strips, series compliance and muscle fiber
heterogeneity may affect mechanical properties and CB cycling kinetics.
It is important to note that experiments designed to apply the
principles of Huxley's theory were performed on isolated frog
sartorius muscles and not on isolated fibers (17).
Huxley's theoretical data (17) were fitted by means of
Hill's data (15) obtained from muscle strips and not from isolated fibers. The equations can, therefore, be applied to
multicellular preparations such as diaphragm muscle strips.
Importantly, four MHC isoforms have been identified in the hindlimb
muscle of frogs (28). Therefore, the model accurately fits
the mechanical properties of a muscle whose fiber composition includes
different MHCs (17). In heterogeneous muscle, the
F-V characteristics are thought to reflect the relative
contribution of each fiber type (48). Likewise, according
to the Huxley equations (17), CB characteristics are thought to reflect the average value of the myosin molecular motors. Therefore, in our study, the CB characteristics of the rat diaphragm strip probably reflected the mean CB behavior of the different MHCs, mainly MHCneo and MHC2A in day
3; MHCneo, MHCslow, and
MHC2A in day 10; MHC2A,
MHC2X, and MHCslow in day 17; and
MHC2A, MHC2X, MHC2B, and
MHCslow in adult rats (47, 49). Moreover,
coexpression of MHC isoforms is not entirely restricted to the early
postnatal period, and, in adult rat diaphragm muscle, ~14% of all
fibers coexpress MHC isoforms (40). Thus, even when
studies are performed at the single-fiber level, it does not totally
resolve the problem related to the coexpression of multiple isoforms of
MHC. In addition, when the specific force of diaphragm muscle fibers is
corrected for the estimated MHC content per half sarcomere, specific
force of fibers expressing different MHC isoforms is comparable
(39). These results indicate that the force per CB is
similar across MHC isoforms and that the difference in specific force
reflects differences in MHC content per half sarcomere. Our results are in agreement with this assumption because we observed that, during postnatal development, there was no increase in the unitary force per
CB but that there was an increase in the total number of CBs, which may
reflect the increase in MHC content per half sarcomere.
In conclusion, in isolated rat diaphragm muscle, we have found that
postnatal maturation was associated with an improved diaphragm contractility and relaxation. The increase in the number of CBs paralleled the postnatal improvement in diaphragm force generation. There were also important changes in CB kinetics during postnatal maturation, but the average force produced by a single CB and the peak
mechanical efficiency remained unchanged. By comparing the rat and the
hamster, it appears that there are few species differences in the
postnatal maturation processes of the diaphragm.
| |
ACKNOWLEDGEMENTS |
The authors would like to thank Dr. D. J. Baker, Fellow of the
Royal College of Anaesthetist, Service d'Aide Médicale Urgent de Paris, Groupe Hospitalier Necker-Enfants Malades) for kindly reviewing the manuscript.
| |
FOOTNOTES |
This work was supported by grants from the Société
Française d'Anesthésie et de Réanimation (Paris,
France) and the Association Française contre la Myopathie (Paris, France).
Address for reprint requests and other correspondence: G. Orliaguet, Département d'Anesthésie-Réanimation,
Groupe Hospitalier Necker Enfants Malades, 149 rue de Sèvres,
75743 Paris Cedex 15, France (E-mail: gorlia{at}club-internet.fr or
gilles.orliaguet{at}nck.ap-hop-paris.fr).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/japplphysiol.00613.2001
Received 14 June 2001; accepted in final form 9 November 2001.
| |
REFERENCES |
1.
Barany, M.
ATPase activity of myosin correlates with speed of muscle shortening.
J Gen Physiol
50:
197-218,
1967[Abstract/Free Full Text].
2.
Belcastro, AN.
Myofibril and sarcoplasmic reticulum changes during muscle development: activity vs. inactivity.
Int J Biochem
19:
945-948,
1987[Web of Science][Medline].
3.
Bertocchini, F,
Ovitt C,
Conti A,
Barone V,
Scholer HR,
Bottinelli R,
Reggiani C,
and
Sorrentino V.
Requirement for the ryanodine receptor type 3 for efficient contraction in neonatal skeletal muscles.
EMBO J
16:
6956-6963,
1997[Web of Science][Medline].
4.
Brutsaert, DL,
Claes VA,
and
Goethal MA.
Effects of calcium on force-velocity-length relations of heart muscle of the cat.
Circ Res
32:
385-392,
1973[Abstract/Free Full Text].
5.
Coirault, C,
Attal P,
Blanc FX,
Chemla D,
and
Lecarpentier Y.
Cross-bridge kinetics in fatigued mouse diaphragm.
Eur Respir J
13:
1055-1061,
1999[Abstract].
6.
Coirault, C,
Chemla D,
and
Lecarpentier Y.
Relaxation of diaphragm muscle.
J Appl Physiol
87:
1243-1252,
1999[Abstract/Free Full Text].
7.
Coirault, C,
Chemla D,
Pery N,
Suard I,
and
Lecarpentier Y.
Mechanical determinants of isotonic relaxation in isolated diaphragm muscle.
J Appl Physiol
75:
2265-2272,
1993[Abstract/Free Full Text].
8.
Coirault, C,
Lambert F,
Joseph T,
Blanc F,
Chemla D,
and
Lecarpentier Y.
Developmental changes in crossbridge properties and myosin isoforms in hamster diaphragm.
Am J Crit Care
156:
959-967,
1997.
9.
D'Albis, A,
Janmot C,
and
Couteaux R.
Species- and muscle type-dependence of perinatal isomyosin transition.
Int J Dev Biol
35:
53-56,
1991[Medline].
10.
Dominguez, R,
Freyzon Y,
Trybus KM,
and
Cohen C.
Crystal structure of a vertebrate smooth muscle myosin motor domain and its complex with the essential light chain: visualization of the pre-power stroke state.
Cell
94:
559-571,
1998[Web of Science][Medline].
11.
Finer, JT,
Simmons RM,
and
Spudich JA.
Single myosin molecule mechanics: piconewton force and nanometre steps.
Nature
368:
113-119,
1994[Medline].
12.
Fratacci, MD,
Levame M,
Rauss A,
Bousbaa H,
and
Atlan G.
Rat diaphragm during postnatal development. I. Changes in distribution of muscle fibre type and in oxidative potential.
Reprod Fertil Dev
8:
391-398,
1996[Medline].
13.
Geiger, PC,
Cody MJ,
Macken RL,
Bayrd ME,
Fang YH,
and
Sieck GC.
Mechanisms underlying increased force generation by rat diaphragm muscle fibers during development.
J Appl Physiol
90:
380-388,
2001[Abstract/Free Full Text].
14.
Gosselin, LE,
Martinez DA,
Vailas AC,
and
Sieck GC.
Interstitial space and collagen alterations of the developing rat diaphragm.
J Appl Physiol
74:
2450-2455,
1993[Abstract/Free Full Text].
15.
Hill, AV.
The heat of shortening and the dynamic constant of muscle.
Proc R Soc Biol (London)
126:
139-195,
1938.
16.
Houadjeto, M,
Bechet JJ,
and
d'Albis A.
Comparative structural and enzymatic properties of skeletal muscle myosin from neonatal and adult rabbits.
Eur J Biochem
191:
695-700,
1990[Web of Science][Medline].
17.
Huxley, AF.
Muscle structure and theories of contraction.
Prog Biophys Chem
7:
255-318,
1957.
18.
Huxley, AF,
and
Simmons RM.
Proposed mechanism of force generation in striated muscle.
Nature
233:
533-538,
1971[Medline].
19.
Johnson, BD,
Wilson LE,
Zhan WZ,
Watchko JF,
Daood MJ,
and
Sieck GC.
Contractile properties of the developing diaphragm correlate with myosin heavy chain phenotype.
J Appl Physiol
77:
481-487,
1994[Abstract/Free Full Text].
20.
Kelly, AM,
Rosser BW,
Hoffman R,
Panettieri RA,
Schiaffino S,
Rubinstein NA,
and
Nemeth PM.
Metabolic and contractile protein expression in developing rat diaphragm muscle.
J Neurosci
11:
1231-1242,
1991[Abstract].
21.
Kyselovic, J,
Leddy JJ,
Ray A,
Wigle J,
and
Tuana BS.
Temporal differences in the induction of dihydropyridine receptor subunits and ryanodine receptors during skeletal muscle development.
J Biol Chem
269:
21770-21777,
1994[Abstract/Free Full Text].
22.
LaFramboise, WA,
Daood MJ,
Guthrie RD,
Butler-Browne GS,
Whalen RG,
and
Ontell M.
Myosin isoforms in neonatal rat extensor digitorum longis, diaphragm, and soleus muscles.
Am J Physiol Lung Cell Mol Physiol
259:
L116-L122,
1990[Abstract/Free Full Text].
23.
Langeron, O,
Coirault C,
Fratea S,
Orliaguet G,
Coriat P,
and
Riou B.
Effects of dantrolene on the diaphragm muscle of the normal and myopathic hamster.
Br J Anaesth
81:
553-555,
1998[Abstract/Free Full Text].
24.
Lecarpentier, Y,
Chemla D,
Blanc FX,
Pourny JC,
Joseph T,
Riou B,
and
Coirault C.
Mechanics, energetics, and crossbridge kinetics of rabbit diaphragm during congestive heart failure.
FASEB J
12:
981-989,
1998[Abstract/Free Full Text].
25.
Lecarpentier, Y,
Coirault C,
Lerebours G,
Desche P,
Scalbert E,
Lambert F,
and
Chemla D.
Effects of angiotensin converting enzyme inhibition on crossbridge properties of diaphragm in cardiomyopathic hamsters of the dilated bio 53-58 strain.
Am J Respir Crit Care Med
155:
630-636,
1997[Abstract].
26.
Lecarpentier, Y,
Coirault C,
Riou B,
Chemla D,
and
Mercadier JJ.
Diaphragm strength and cross-bridge properties during chronic growth hormone hypersecretion.
Eur Respir J
13:
1070-1077,
1999[Abstract].
27.
Lloyd, JS,
Brozanski BS,
Daood M,
and
Watchko JF.
Developmental transitions in the myosin heavy chain phenotype of human respiratory muscle.
Biol Neonate
69:
67-75,
1996[Web of Science][Medline].
28.
Lutz, GJ,
Cuizon DB,
Ryan AF,
and
Lieber RL.
Four novel myosin heavy chain transcripts define a molecular basis for muscle fibre types in Rana pipiens.
J Physiol (Lond)
508:
667-680,
1998[Abstract/Free Full Text].
29.
Martin-Caraballo, M,
Campagnaro PA,
Gao Y,
and
Greer JJ.
Contractile and fatigue properties of the rat diaphragm musculature during the perinatal period.
J Appl Physiol
88:
573-580,
2000[Abstract/Free Full Text].
30.
Maxwell, LC,
McCarter RJM,
Kuehl TJ,
and
Robotham JL.
Development of histochemical and functional properties of baboon respiratory muscles.
J Appl Physiol
54:
551-561,
1983[Abstract/Free Full Text].
31.
Moore, BJ,
Feldman HA,
and
Reid MB.
Developmental changes in diaphragm contractile properties.
J Appl Physiol
75:
522-526,
1993[Abstract/Free Full Text].
32.
Nishizaka, T,
Miyata H,
Yoshikawa H,
Ishiwata S,
and
Kinosita K, Jr.
Unbinding force of a single motor molecule of muscle measured using optical tweezers.
Nature
377:
251-254,
1995[Medline].
33.
Orliaguet, G,
Langeron O,
Coirault C,
Fratea S,
Coriat P,
and
Riou B.
Effects of dantrolene on rat diaphragm muscle during postnatal maturation.
Anesthesiology
94:
468-474,
2001[Web of Science][Medline].
34.
Powers, SK,
Criswell D,
Herb RA,
Demirel H,
and
Dodd S.
Age-related increases in diaphragmatic maximal shortening velocity.
J Appl Physiol
80:
445-451,
1996[Abstract/Free Full Text].
35.
Rayment, I,
Rypniewski WR,
Schmidt-Base K,
Smith R,
Tomchick DR,
Benning MM,
Winkelmann DA,
Wesenberg G,
and
Holden HM.
Three-dimensional structure of myosin subfragment-1: a molecular motor.
Science
261:
50-58,
1993[Abstract/Free Full Text].
36.
Sheterline, P,
Clayton J,
and
Sparrow JC.
Protein profile.
In: Actins (3rd ed.). London: Academic, 1996, p. 1-76.
37.
Sieck, GC,
Cheung TS,
and
Blanco CE.
Diaphragm capillarity and oxidative capacity during postnatal development.
J Appl Physiol
70:
103-11,
1991[Abstract/Free Full Text].
38.
Sieck, GC,
Fournier M,
and
Blanco CE.
Diaphragm muscle fatigue resistance during postnatal development.
J Appl Physiol
71:
458-464,
1991[Abstract/Free Full Text].
39.
Sieck, GC,
Han YS,
Prakash YS,
and
Jones KA.
Cross-bridge cycling kinetics, actomyosin ATPase activity and myosin heavy chain isoforms in skeletal and smooth respiratory muscles.
Comp Biochem Physiol B Biochem Mol Biol
119:
435-450,
1998[Medline].
40.
Sieck, GC,
and
Prakash YS.
Cross-bridge kinetics in respiratory muscles.
Eur Respir J
10:
2147-2158,
1997[Abstract].
41.
Smith, D,
Green H,
Thomson J,
and
Sharatt M.
Capillary and size interrelationships in developing rat diaphragm, EDL, and soleus muscle fiber types.
Am J Physiol Cell Physiol
256:
C50-C58,
1989[Abstract/Free Full Text].
42.
Suga, H,
Goto Y,
Igarashi Y,
Yasumura Y,
Nozawa T,
Futaki S,
and
Tanaka N.
Cardiac cooling increases Emax without affecting relation between O2 consumption and systolic pressure-volume area in dog left ventricle.
Circ Res
63:
61-71,
1988[Abstract/Free Full Text].
43.
Tanaka, H,
and
Shigenobu K.
Effect of ryanodine on neonatal and adult rat heart: developmental increase in sarcoplasmic reticulum function.
J Mol Cell Cardiol
21:
1305-1313,
1989[Web of Science][Medline].
44.
Tarroni, P,
Rossi D,
Conti A,
and
Sorrentino V.
Expression of the ryanodine receptor type 3 calcium release channel during development and differentiation of mammalian skeletal muscle cells.
J Biol Chem
272:
19808-19813,
1997[Abstract/Free Full Text].
45.
Trang, TT,
Viires N,
and
Aubier M.
In vitro functions of the rat diaphragm during postnatal development.
J Dev Physiol
17:
1-6,
1992[Web of Science][Medline].
46.
Watchko, JF,
Brozanski BS,
O'Day TL,
Guthrie RD,
and
Sieck GC.
Contractile properties of the rat external abdominal oblique and diaphragm muscles during development.
J Appl Physiol
72:
1432-1436,
1992[Abstract/Free Full Text].
47.
Watchko, JF,
Daod MJ,
and
Sieck GC.
Myosin heavy chain transitions during development. Functional implications for the respiratory musculature.
Comp Biochem Physiol A Physiol
119:
459-470,
1998.
48.
Woledge, RC,
Curtin NA,
and
Homster E.
Energetic aspects of muscle contraction.
Monogr Physiol Soc
41:
27-117,
1985.
49.
Zhan, WZ,
Watchko JF,
Prakash YS,
and
Sieck GC.
Isotonic contractile and fatigue properties of developing rat diaphragm muscle.
J Appl Physiol
84:
1260-1268,
1998[Abstract/Free Full Text].
J APPL PHYSIOL 92(3):1074-1082
8750-7587/02 $5.00
Copyright © 2002 the American Physiological Society
TOP
ABSTRACT
INTRODUCTION
![<IT>×</IT>[g<SUB>1</SUB><IT>+</IT>f<SUB>1</SUB><IT>V×ϕ</IT><SUP><IT>−</IT>1</SUP>(1<IT>−</IT>e<SUP><IT>−ϕ/V</IT></SUP>)]](/content/vol92/issue3/fulltext/1074/img004.gif)
![[1<IT>+</IT>(f<SUB>1</SUB><IT>+</IT>g<SUB>1</SUB>)<SUP>2</SUP><IT>·</IT>g<SUP><IT>−</IT>2</SUP><SUB>2</SUB><IT>·V/</IT>(2<IT>ϕ</IT>)]}](/content/vol92/issue3/fulltext/1074/img013.gif)
