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Department of Human Anatomy and Physiology, University College, Dublin 2, Ireland
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
ABSTRACT 
McGuire, Michelle, Michael F. Carey, and John J. O'Connor.
Almitrine and doxapram decrease fatigue and increase subsequent recovery in isolated rat diaphragm. J. Appl.
Physiol. 83(1): 52-58, 1997.
The effects of
almitrine bimesylate and doxapram HCl on isometric force produced by in
vitro rat diaphragm were studied during direct muscle activation at
37°C. Doxapram and almitrine ameliorate respiratory failure
clinically by indirectly increasing phrenic nerve activity. This study
was carried out to investigate possible direct actions of these agents
on the diaphragm before and after fatigue of the fibers. Two age groups
of animals were chosen [6-14 wk (group
1) and 50-55 wk (group
2)] because it is known that increasing age
decreases a muscle fiber's resistance to fatigue. Muscle strips were
isolated from both group 1 and group 2 and directly stimulated (2-ms
pulse duration, 5-15 V) to produce twitch tensions of 1.3 and 2.1 N/cm2, respectively. At low
concentrations, doxapram (
20 µg/ml) and almitrine (12 µg/ml)
had no effect on twitch contraction or 100-Hz tetanic tension. However,
40 µg/ml doxapram and 30 µg/ml almitrine increased twitch tension
by 9.0 ± 1.4 and 11.6 ± 1.9%, respectively, in animals of
group 2 (n = 5). A fatigue protocol consisting
of low-frequency stimulation (30-Hz trains, 250-ms duration every 2 s
for 5 min) caused a reduction of twitch tension in animals of
group 1 (48 ± 4% of
control) and group 2 (28 ± 4% of
control). At 90 min postfatigue, the twitch tension recovered to 72 ± 3 and 42 ± 2% of control values in
group 1 and group
2, respectively. In the presence of doxapram (20 µg/ml), there was a significant increase in the recovery of twitch
tension at 90 min in group 1 and
group 2 (84.5 ± 3.2 and 80.1 ± 2.8%, respectively) compared with controls at 90 min postfatigue. In
the presence of almitrine (12 µg/ml), there was a full recovery from
fatigue in group 1 animals (100% of
control) and a recovery to 95.6 ± 2.1% of control in
group 2 animals at 90 min. These
results demonstrate a significant improvement in the rapidity and
magnitude of recovery from fatigue in the rat diaphragm muscle in the
presence of both doxapram and, especially, almitrine. These effects may
be due to changes in intracellular calcium, ADP/ATP ratios, or oxygen
free radical scavenging.
almitrine bimesylate; doxapram hydrochloride; muscle fatigue; direct stimulation
INTRODUCTION RESPIRATORY MUSCLE FATIGUE is well recognized as one of
the major causes of respiratory failure (5), and many pharmacological agents have been used in the treatment of this condition with variable
success. Diaphragmatic contractility has been reported to
be increased by aminophylline, which also increases phrenic nerve
activity (2, 3, 9). The peripheral chemoreceptor stimulators almitrine
and doxapram have also been used to increase ventilation in respiratory
failure by indirectly increasing efferent phrenic nerve activity (19).
It seems illogical that using these agents to increase phrenic nerve
activity to an already fatigued diaphragm should increase diaphragmatic
performance. The fact that this does happen in some cases prompted us
to study the effects of almitrine and doxapram on fatigued diaphragm in
vitro.
It has been recently shown that prolonged periods of muscular ischemia
and deoxygenation can induce muscle injury that is free radical
mediated (1, 25, 31). Also, during repetitive contraction of muscle
fibers, there is an increase in free radical concentration in the
myocyte (26, 27). Almitrine has recently been shown to have oxygen free
radical-scavenging ability (6, 7). It also increases calcium release
from the sarcoplasmic reticulum and is used in the treatment of
respiratory failure (32). At present, however, the basic mechanisms of
action of almitrine are unknown. Doxapram is another compound reported
to be useful in the treatment of respiratory failure (4, 23), although
its direct mode of action on myocytes is not well documented.
The rat diaphragm is particularly resistant to fatigue because of its
mixed composition of fiber types, namely, 27% fast twitch, glycolytic;
39% slow twitch, oxidative; and 34% fast twitch, oxidative, glycolytic (see Ref. 30). This mixed fiber type ratio is reasonably similar to that found in humans, with shortening velocities and adenosinetriphosphatase activity similar to those of myosin
(30). However, with repeated stimulation, the diaphragm
becomes fatigued (fast-twitch glycolytic fibers have the lowest
resistance to fatigue), and its ability to develop tension is reduced
(29).
It has previously been demonstrated that many biochemical and
morphological changes occur in skeletal muscle with aging (13). Increasing age also decreases the maximum tension developed by the
diaphragm, and the older diaphragm has less resistance to fatigue (21,
33). In this study, a low-frequency protocol was used to give a
reproducible fatigue and recovery profile in diaphragm muscle strips
from young (6-14 wk) and more mature animals (50-55 wk; Ref.
24). The effects of almitrine and doxapram on this profile were then
investigated.
METHODS
) and those treated with 2 (
),
10 (
), and 20 (
) µg/ml doxapram. There was a 72% recovery at
90 min after fatigue run in control animals. Doxapram at concentrations
of 10 and 20 µg/ml significantly increased recovery postfatigue
(P < 0.01 and
P < 0.001, respectively; Fisher's
test). B: time course of recovery of
the twitch tension from fatigue over 90 min in group
2 control animals () and those treated with 2 (),
10 (), and 20 () µg/ml doxapram. There was a 41.5% recovery at
90 min after fatigue run in control animals. Doxapram at concentrations
of 10 and 20 µg/ml significantly increased recovery postfatigue
(P < 0.001; Fisher's test). Each
point is mean ± SE; n = 5 observations except for controls (n = 10). Twitch tension is expressed as percentage of prefatigue control.
) and
those treated with 3 (), 12 (), and 30 () µg/ml almitrine. There was a 71% recovery at 90 min after fatigue run in control animals. All concentrations of almitrine gave rise to 100% recovery from fatigue (P < 0.001 for all;
Fisher's test). B: time course of
recovery of twitch tension from fatigue run over 90 min in group 2 control animals () and
those treated with 3 (), 12 (), and 30 () µg/ml almitrine.
There was a 40.4% recovery at 90 min after fatigue run in control
animals. There was a recovery of 95.6, 100, and 100% at concentrations
of 3, 12, and 30 µg/ml respectively compared with prefatigue controls
(P < 0.01; Fisher's test). Each point is mean ± SE;
n = 5 observations except for controls
(n = 10). Twitch tension is expressed
as percentage of prefatigue control.
RESULTS
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Figure 2 shows the twitch tension (%) after the fatigue protocol compared with prefatigue values for both doxapram (A)- and almitrine (B)- treated animals in group 1 and group 2. Application of 2 and 10 µg/ml doxapram had no significant effect on the twitch tension postfatigue in group 1 and group 2 (n = 5; Fig. 2A). However, a concentration of 20 µg/ml doxapram significantly increased twitch tension postfatigue compared with postfatigue controls in both groups of animals (Fig. 2A; P < 0.01; n = 5 for both). Almitrine at a concentration of 3 µg/ml had no effect on twitch tension postfatigue in group 1 and group 2 compared with postfatigue controls (Fig. 2B). However, 12 and 30 µg/ml almitrine increased twitch tension postfatigue in both groups (Fig. 2B; P < 0.01; n = 5 for both group 1 and group 2 at both concentrations). Recovery of twitch tension after the fatigue protocol. In control group 1 animals the twitch tension recovered to 72 ± 3.4% (compared with controls prefatigue), 90 min after the fatigue run (n = 8; Fig. 3A). In group 2, twitch tension recovered to 41.5 ± 1.8% after 90 min (n = 8; Fig. 3B). In a separate set of two experiments, twitch tension recovered to 94% after 180 min in group 1 and to 86% control after 210 min in group 2, which indicated that near full recovery was obtainable in both group of muscle fibers after a longer recovery time. In the doxapram-treated group 1 animals, concentrations of 2, 10, and 20 µg/ml gave rise to recoveries of 77.5 ± 2.5, 79.9 ± 2.7, and 84.5 ± 3.2% of control, respectively, at 90 min (n = 5; Fig 3A; the latter 2 doses being significantly different from controls; Fisher's test post-ANOVA; P < 0.01 and P < 0.001, respectively). Doxapram-treated group 2 animals at doses of 2, 10, and 20 µg/ml had recoveries of 45.5 ± 2.3, 65.1 ± 2.7, and 80.1 ± 2.8% of control, respectively, at 90 min (the 2 higher doses being significantly different from controls; Fisher's test post-ANOVA; P < 0.001 for both; n = 5; Fig. 3B). In group 1 animals treated with almitrine (3, 12, and 30 µg/ml), there was a 100% recovery of twitch tension at all concentrations (n = 5; Fisher's test post-ANOVA; P < 0.001 for all concentrations; Fig. 4A). In group 2 almitrine-treated animals, recovery postfatigue was 95.6 ± 2.1, 100, and 100% for concentrations of 3, 12, and 30 µg/ml, respectively (Fig. 4B; n = 5; P < 0.001 for all concentrations).
DISCUSSION
The present data show that almitrine and doxapram, at concentrations that either had no effect on twitch tension or did have a significant enhancing effect on twitch tension, were able to significantly improve recovery from fatigue in young and mature animals. In the case of almitrine there was a complete and rapid recovery from fatigue. The results of this study also confirm previous work in the baboon (21) and hamster (33), which showed that the respiratory muscles of younger animals have a higher resistance to fatigue than do those of older animals. This may be due to the greater number of mitochondria-rich and/or highly oxidative diaphragm muscle fibers in the younger animals (13).
Our studies gave rise to Po values in mature animals similar to those previously reported (e.g., Ref. 1). However, our Po values for young animals were significantly lower. This may have been due in part to the use of two animal groups with significantly different body weight, different muscle fiber composition (13), different histochemical structure (21), and nonuniformity of bundle weight and Lo and to the assumption of a similar muscle density (1.056 g/cm3) to obtain CSAs for both groups of animals. It is unlikely, however, that these differences alone can account for this discrepancy.
The fatigue protocol in this study is of a low-frequency type similar to that used by Reid et al. (26). This form of fatigue has been shown to mimic the metabolic changes produced by peripheral fatigue in vivo, i.e., depletion of glycogen stores and phosphocreatine levels as well as a drop in intracellular pH and a rise in intracellular phosphate (Pi). It is thought that this acidosis and increased Pi inhibit the actin-myosin interaction giving rise to contractile failure. Reactive oxygen intermediates seemed to be implicated in this form of fatigue (25, 26). Rapid- and slow-onset fatigue protocols in the isolated rat diaphragm have also been documented by Kolbeck and Nosek (17). High-frequency fatigue protocols seem to bring about an impulse-propagation block across the sarcolemma and do not seem to involve reactive oxygen intermediates (25). Rochester (29) suggested that the maximum recovery from high-frequency fatigue is relatively fast (~30 min), compared with maximum recovery from low-frequency fatigue, which does not occur until at least 1 h or more has elapsed after the fatigue. The results of this study tend to agree with this observation, namely, the younger animals reach a peak recovery after 180 min and the older animals after 210 min. This prolonged time period necessary for recovery may imply that damage occurs to subcellular organelles or membranes, although the high recovery rates suggest that this damage is minimal. Also, it is known that fatiguing muscle fibers undergo metabolite accumulation and loss of calcium homeostasis. This may directly inhibit contractile protein interactions and thereby depress muscle function especially in the mature muscle fibers (25). The larger CSA of the mature muscle fibers must also be taken into account, whereby oxygen diffusion may be impaired to a greater extent. The in vitro preparation used in this study would not have been able to replenish the muscle fiber energy requirements or take away metabolites to the same extent as the in vivo diaphragm. Nevertheless, we feel that the very large effect of almitrine (100% recovery of the twitch tension in many cases) indicates that little damage may have occurred to the individual fibers.
Doxapram and almitrine have previously been used in the treatment of respiratory failure. Doxapram increases central respiratory drive by stimulating peripheral arterial chemoreceptors in addition to central respiratory neurons (23). Almitrine is known to act via the peripheral arterial chemoreceptors, raising carotid sinus nerve output and minute ventilation (4, 19). It also improves gas exchange by enhancing hypoxic pulmonary vasoconstriction (14). The in vitro preparation in these studies would seem to exclude many of these effects.
The mechanism by which doxapram affects the directly stimulated diaphragm in our experiments is unclear. It has been tested on the neuromuscular junction in the rat phrenic nerve diaphragm preparation used by Pollard et al. (23), who found that it has a presynaptic facilitatory effect. However, in the presence of partial neuromuscular block, it produces a seemingly postjunctional inhibitory effect, albeit only when it is given at very high doses. Because the direct diaphragm stimulation in this study is independent of neuromuscular junction activity, our results would seem to be in contrast to the work of Pollard et al. However, it is interesting to note that at very high doses (40 µg/ml) doxapram did enhance baseline twitch and tetanic tension.
Almitrine may act like the agent aminophylline, a compound also reported to improve diaphragmatic contractility (2, 3, 18) and reverse diaphragmatic fatigue (12). It is thought that aminophylline increases diaphragmatic contractility by increasing intracellular calcium concentration (9) as well as changing high-energy Pi metabolism (15). Almitrine and doxapram may also increase intracellular calcium by either increasing calcium flow into the cell or preventing its uptake by the sarcoplasmic reticulum. Indeed, in our laboratories it was found that almitrine attenuates the inhibitory effect of dantrolene on twitch tension (unpublished observations). Fitts (11), in a recent comprehensive review, has focused attention on the mechanism of muscle fatigue at the cellular level. Recent studies have shown that almitrine can act directly on mitochondria, decreasing the cytosolic and mitochondrial ATP/ADP ratios and thus decreasing ATP utilization (20). Our present set of experiments provided no direct proof for these mechanisms of action. There is also increasing evidence that an increased production of reactive oxidant species occurs in exercising diaphragmatic muscle (10, 25). Reid et al. (26) postulate that these species contribute to muscular fatigue in vitro (see also Ref. 10). Also, muscles that produce the most reactive oxidant species also show the greatest fatigue (27).
Almitrine has also been shown to reduce the adverse effects of cerebral ischemia in cats (7), which may be due to an oxygen free radical-scavenging effect (6, 8). Therefore, it would be of interest to discover whether almitrine had a similar mode of action in the fatigued diaphragm in our studies. Muscular fatigue has been shown to be attenuated by pretreatment with antioxidants such as N-acetylcysteine (NAC; 16, 28). In our laboratories it was found that the effects of NAC are not additive to those of almitrine on muscle fatigue (unpublished observations). The importance of these cellular effects of almitrine on diaphragm muscle fatigue remains to be established. Whether they can be attributed to the greater effect (decrease in time to recovery and increase in magnitude of recovery) of almitrine on fatigued muscle compared with doxapram is still unknown.
In summary, almitrine and doxapram, at doses that had no effect on baseline muscle contractility, both decreased the time to maximum recovery and increased the percent recovery from fatigue (in the case of almitrine to 100% of control). These significant effects may be due to increased intracellular calcium, altered ADP/ATP ratios (20), or oxygen free radical scavenging.
ACKNOWLEDGEMENTS
We thank Servier Ireland for the kind gift of almitrine.
FOOTNOTES
Address for reprint requests: J. O'Connor, Dept. of Human Anatomy and Physiology, Univ. College, Earlsfort Terrace, Dublin 2, Ireland (E-mail: John.OConnor@UCD.IE).
Received 5 August 1996; accepted in final form 26 February 1997.
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