Departments of Physiology (H.G.-S., R.C.) and Pharmacology and
Experimental Therapeutics (E.F.R.P., E.X.A.), University of Maryland
School of Medicine, Baltimore, Maryland; and Departamento de
Farmacologia Básica e Clínica (E.X.A., N.G.C., Y.A.,
P.A.M.), Instituto de Ciências Biomédicas, and Departamento
de Fármacos (P.C.L., C.A.M.F., E.J.B.), Faculdade de
Farmácia, Centro de Ciências da Saúde, Universidade
Federal do Rio de Janeiro, Rio de Janeiro, Brazil
This study was designed to investigate the effects on single skeletal
muscle fibers of a novel thienylhydrazone, referred to as LASSBio-294,
which is a bioisoster of pyridazinone compounds that inhibit the cyclic
AMP-specific phosphodiesterase (PDE) 4. Twitch and fatigue were
analyzed in single skeletal muscle fibers isolated from either the
semitendinous or the tibialis anterior muscles dissected from the frog
Rana pipiens. LASSBio-294 (12.5-100 µM) increased
twitch tension, accelerated the maximal rate of tension decay during
relaxation, and had very little effect in the maximal rate of tension
development of muscle fibers directly stimulated at 
30 Hz. The
positive inotropic effect of LASSBio-294 developed slowly, reaching its
maximum at 40 min and was inversely proportional to the frequency of
stimulation, becoming negligible at 60 and 90 Hz. The
concentration-response relationship for LASSBio-294-induced potentiation of twitch tension was bell-shaped, with maximal effect occurring at 25 µM. In addition, LASSBio-294 reduced development of
fatigue induced by tetanic stimulation of the muscle fibers and reduced
the time needed for 80% prefatigue tension recovery after fatigue had
developed to 50% of the maximal pretetanic force. These effects of
LASSBio-294 can be fully explained by stimulation of the sarcoplasmic
reticulum Ca2+ pump and could be ascribed to an
increase in cellular levels of cyclic AMP due to PDE inhibition. The
novel thienylhydrazone LASSBio-294 may be useful for treatment of
patients suffering from conditions in which muscle fatigue is a
debilitating symptom (e.g., chronic heart failure).
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Introduction |
Exertional
skeletal muscle fatigue is a major debilitating symptom in patients
suffering from a number of chronic disorders, including chronic heart
failure (CHF) (Wilson, 1995
, and references therein). Skeletal muscle
fatigue develops gradually during all forms of exercise (for review,
see Jones and Killian, 2000); however, it does so more rapidly in
patients with CHF than in normal subjects (for review, see Bishop et
al., 1998; Lunde et al., 1998). The enhanced skeletal muscle fatigue in
patients with CHF is not due to impaired control of motor drive by the
central nervous system or to dysfunctions in the neuromuscular
transmission; rather, it is caused by intrinsic physiological changes
in excitation-contraction (e-c) coupling in the skeletal muscle cells
themselves (Perreault et al., 1993; Wilson, 1995; Bishop et al., 1998).
Skeletal and heart muscles share a number of properties, including
regulation of contractile function by cyclic AMP (Gonzalez-Serratos et
al., 1981), and evidence has been provided to support a unifying hypothesis that changes in heart and skeletal muscle inotropism associated with CHF are related to altered cyclic AMP content and/or
responsiveness in the myocyte (Morgan, 1991; Perreault et al., 1993;
Bishop et al., 1998). In fact, drugs that increase cytoplasmic levels
of cyclic AMP, by either blocking its degradation or increasing its
production, are recognized not only for their positive inotropic
effects in cardiac and skeletal muscles (Gonzalez-Serratos et al.,
1981; Francis et al., 2001) but also for their ability to reduce
skeletal muscle fatigue (Bishop et al., 1998; Fujii et al., 1998).
A novel thienylhydrazone compound
(2'-thienylidene)3,4-methylenedioxybenzoylhydrazine, herein referred to
as LASSBio-294, was initially obtained as part of a program of
synthesis of novel anti-inflammatory leads with an
N-acylhydrazone skeleton (Figueiredo et al., 2000).
Subsequently, this compound was identified as a bioisoster of a family
of pyridazinone compounds that increase cyclic AMP levels by inhibiting
selectively the cyclic AMP-specific, low-Km PDE4 (Piaz et al., 1997). The
present study was undertaken to investigate the effects of this novel
thienylhydrazone in the contractile properties of single skeletal
muscle fibers of the frog.
In addition to demonstrating that LASSBio-294 has positive inotropic
effects in single fibers of phasic muscles of the frog, the results
presented herein also show that the compound reduces fatigue
development and accelerates recovery of maximal tetanic force after
fatigue has developed. Some lines of evidence suggest that the effects
of LASSBio-294 on skeletal muscle inotropism and fatigue can be
accounted for by PDE inhibition. Thus, LASSBio-294, by virtue of its
ability to increase force of contraction and decrease fatigue of
skeletal muscles, can become part of the therapeutic interventions
designed to decrease exertional fatigue, and, consequently, improve the
quality of life of patients suffering from CHF (LeJemtel et al., 1986)
and other physiopathological conditions in which skeletal muscle
dysfunctions are associated with decreased levels of cytosolic cyclic AMP.
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Materials and Methods |
Preparations.
Single skeletal muscle fibers were freshly
isolated from either the semitendinous or the tibialis anterior muscles
dissected from the frog Rana pipiens as previously described
(Gonzales-Serratos et al., 1981). After isolation, muscle cells
remained in the dissecting dish for at least 30 min in Ringer's
solution, which consisted of 115 mM NaCl, 2.5 mM KCl, 1.8 mM
CaCl2, 0.2 mM MgCl2 (pH was adjusted with phosphates to 7.2). Next, the fibers were stimulated with
single low-voltage electrical shocks. If they responded with brisk
twitches and there were no signs of membrane damage, the fibers were
used; otherwise, they were discarded. The sartorius muscle was also
dissected out from R. pipiens for recordings of membrane and
action potentials (see below). All experiments were carried out at room
temperature (20-22°C).
Muscle Twitch and Fatigue.
Each single fiber was transferred
to the experimental chamber, which consisted of a 0.3-ml narrow channel
where solutions could be changed several times within 5 s. In the
chamber, one tendon was gripped with a small clamp while the other
tendon was attached to the hook of an Ekharts type force transducer
(Senso Nor, Horten, Norway). The stimulating electrode consisted of
platinum wires placed on each side of the fiber. The muscle fiber was
then stretched 1.3 times its slack length to reach an average sarcomere length of approximately 2.6 µm. Subsequently, the fiber was
stimulated with single electrical pulses of 0.5-ms duration with
variable voltage. The voltage was steadily increased until the
threshold for contraction was reached. This voltage was then increased
by 50%, and the experimental protocol was started.
The stimulating protocol consisted of a series of single twitches
elicited every 3 s until the peak twitch force was the same for
five consecutive times. The twitches were followed by different frequencies of tetanic stimulation of 10, 30, 60, and 90 Hz. Each fiber
rested for 3 min between each of the tetanic stimulations. When the
whole series of stimulations was repeated, the fiber was allowed to
rest for 10 min between each series of twitches and tetanic
stimulations. Fatigue was induced by repetitive cycles of electrical
stimulation. Each cycle consisted of a train of electrical shocks
delivered at 60 Hz for 0.8 s followed by a single twitch after
2.2 s and repeated every 4.75 s.
Intracellular Recordings.
Resting membrane potential and
action potentials were recorded intracellularly during stimulation of
frog sartorius muscles. By means of two fine external electrodes, one
or two fibers were stimulated on the surface of sartorius muscles that
were previously stretched 1.6 times their equilibrium lengths. Under
this condition, it was possible to record membrane potentials from a
stimulated fiber without damaging it during the contraction by using
floating recording microelectrodes (Bezanilla et al., 1972). The
recording microelectrode was connected through a Ag-AgCl electrode to
the input of a cathode follower with negative capacity compensation (World Precision Instruments, Sarasota, FL).
Drugs.
LASSBio-294, 2'-thienylidene
3,4-methylenedioxybenzoylhydrazide or
3,4-methylenedioxybenzoyl-2'-thienylhydrazone or
N-((1E)-1-aza-2-(2-thienyl)vinyl)-2H-benzo[3,4-d]1,3-dioxolan-5-ylcarboxamide, was synthesized as part of a program designed to develop a series of
new N-acylhydrazone derivatives with
anti-inflammatory properties (Figueiredo et al., 2000) in the
Departamento de Fármacos, Faculdade de Farmácia, Centro de
Ciências da Saúde, Universidade Federal do Rio de Janeiro,
Rio de Janeiro, Brazil. Based on principles of nonclassical
bioisosterism, LASSBio-294 was identified as a bioisoster of bioactive
6-aryl-4,5-heterocyclic-fused pyridazinone compounds (Fig.
1A) that are potent and selective
inhibitors of PDE4 (Piaz et al., 1997). Briefly, according to the
procedure described previously (Lima et al., 2000), LASSBio-294 was
synthesized from the starting material safrole
(4-allyl-1,2-methylenedioxybenzene), which is a readily available
natural substance present in sassafras oil (Fig. 1B). The first step in
the synthesis consisted of a base-catalyzed isomerization of safrole
(1) that yielded to isosafrole (2). By oxidative cleavage, isosafrole
was converted into piperonal (3). Then, by using the Yamada's
oxidative procedure, piperonal (3) was "one-pot" converted, with
90% yield, in the methyl 3,4-methylenedioxybenzoate (4). Subsequently,
the key 3,4-methylenedioxyhydrazine (5) was obtained with 70% yield by
means of nucleophilic substitution of the ester (4) with hydrazine
hydrate in ethanol at reflux for 3.5 h. Finally, LASSBio-294 was
obtained with 75% yield by condensing the hydrazine derivative with
equimolar amounts of thiophene-2-carboxaldehyde in ethanol by using
hydrochloric acid as a catalyst.
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Fig. 1.
A, structural similarity between LASSBio-294 and
bioactive pyridazinones. B, schematic representation of the synthetic
route to the novel thienylhydrazone LASSBio-294.
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A stock solution containing LASSBio-294 (50 mM) was obtained by
dissolving the compound in DMSO. A solution containing 100 µM
LASSBio-294 was prepared by diluting the stock solution in Ringer's
solution under sonication. The final test concentrations were obtained
by further diluting the Ringer's solution containing 100 µM
LASSBio-294. Because DMSO was used as a dispersing agent to dissolve
LASSBio-294, the Ringer's solution used under control conditions
contained the same amount of DMSO as that present in the test solutions
with LASSBio-294.
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Results |
Effects of LASSBio-294 on Force of Contraction of Skeletal Muscle
Fibers: Time and Concentration Dependence.
To investigate the
effect of LASSBio-294 on muscle twitch tension, single fibers of the
frog semitendinous or tibialis anterior muscles were directly
stimulated at 0.1 Hz in the absence and in the presence of different
concentrations of the compound. Three minutes after the last control
twitch was recorded, the fibers were perfused continuously with
Ringer's solution containing LASSBio-294 (12.5, 25, 75, or 100 µM),
and twitch tension was analyzed at various times. LASSBio-294 increased
twitch tension (Fig. 2A). The onset of
the positive inotropic effect of LASSBio-294 (25 µM) was observed at
about 5 min after starting perfusion of the muscle fibers with the
drug; the effect was maximal at 40 min after starting perfusion of the
fibers (Fig. 2B).
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Fig. 2.
Effects of LASSBio-294 on twitch tension of single
skeletal muscle fibers. A, twitch tension was recorded from a single
skeletal muscle fiber before (control) and 40 min after perfusion with
Ringer's solution containing LASSBio-294 (25 µM). B, time dependence
of the positive inotropic effect of LASSBio-294 (25 µM). Maximal
twitch tension recorded under control condition
(To) was taken as 1 and used to normalize the
maximal twitch tension (Tn) recorded at various
times in the presence of LASSBio-294. Symbols and bars represent mean
and S.E.M., respectively, of results obtained from three fibers. C,
concentration dependence of the positive inotropic effect of
LASSBio-294. Peak twitch tension (To) measured
prior to exposure of the fibers to a given concentration of LASSBio-294
was taken as 1 and used to normalize the peak twitch tension
(Tn) measured after 40-min exposure of the
fibers to the compound. Symbols and bars represent mean and S.E.M.,
respectively, of results obtained from four experiments.
*p < 0.05 and **p < 0.01, according to the paired Student's t test.
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To determine the magnitude of the positive inotropic effect of
LASSBio-294, the fibers were stimulated at 0.1 Hz before and after
40-min perfusion with solution containing various concentrations of the
drug. The concentration-response relationship for LASSBio-294-induced potentiation of muscle twitch was bell-shaped (Fig. 2C). At 12.5 µM,
LASSBio-294 caused a small, albeit significant increase in twitch
tension. The magnitude of the positive inotropic effect of LASSBio-294
was significantly enhanced upon increasing the concentration of the
drug to 25 µM (Fig. 2C). However, the positive inotropic effect of
LASSBio-294 decreased at concentrations 
50 µM (Fig. 2C).
Positive Inotropic Effect of LASSBio-294 Depends on Frequency of
Stimulation and Is Slowly Reversible.
As indicated above, 40-min
perfusion of single muscle fibers with Ringer's solution containing
LASSBio-294 (12.5 µM) caused a small increase in twitch tension.
However, when the fibers were stimulated at 10 or 30 Hz 17 min after
they had been continuously bathed with LASSBio-294 (12.5 µM), twitch
tension was increased further (Fig. 3, A
and C). The effect of LASSBio-294 (12.5 µM) on maximal twitch tension
decreased as the frequency of stimulation was increased to 60 Hz and
became negligible when the fibers were stimulated at 90 Hz. Similar
effects were observed when the muscle fibers were exposed to 25 µM
LASSBio-294 (Fig. 3, B and C).
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Fig. 3.
Effects of LASSBio-294 on force development of
skeletal muscle fibers stimulated at various frequencies and during
wash. A and B, effects of LASSBio-294 (12.5 and 25 µM) on force
development in single muscle fibers stimulated at 0.1 (single twitch,
st), 10, 30, 60, and 90 Hz, before (control), after 40-min perfusion
with Ringer's solution containing a given concentration of
LASSBio-294, and 17 min after perfusion with drug-free Ringer's
solution (wash). C, effects of LASSBio-294 (12.5 and 25 µM) on
fractional twitch potentiation at the different frequencies of
stimulation. Symbols and bars represent mean and S.E.M. of results
obtained from four experiments. D, plots of fractional twitch tension
calculated from data obtained from muscle fibers during various
recording times under the experimental conditions indicated by the
dotted and continuous lines. Symbols and bars represent mean and
S.E.M., respectively, of results obtained from four fibers. The
calibration bars shown in the top traces A and B apply to the lower
traces of A and B.
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The positive inotropic effect of LASSBio-294 was not easily reversed
upon washing the preparations with drug-free Ringer's solution (Fig.
3, A, B, and D). A 40-min exposure of muscle fibers (n = 3) to LASSBio-294 (25 µM) increased by 35 ± 10% the twitch tension elicited by 0.1-Hz stimulus (Fig. 3D). After a 17-min wash of
the preparation with Ringer's solution and subsequent exposure to
LASSBio-294 (25 µM), twitch tension was 57 ± 14% higher than
that recorded under control condition. This could be explained by a
cumulative effect of the drug. After further washing the preparations
with drug-free solution, there was an additional increase in twitch
tension (Fig. 3D). A subsequent exposure of the washed preparations to
LASSBio-294 (25 µM) reduced twitch tension; however, twitch tension
did not return to control levels. It was still 71 ± 14% larger
than that recorded under control condition, i.e., prior to exposure of
the preparation to the drug (Fig. 3D). Twitch tension increased again
when LASSBio-294 was removed from the recording chamber; now it was
twice as large as that recorded under control condition (Fig. 3D).
Similar results were obtained from fibers perfused with LASSBio-294
(12.5, 50, 75, and 100 µM).
The positive inotropic effect of LASSBio-294 was slowly reversible. In
three different preparations that had been exposed for 40 min to
LASSBio-294 (25 µM) it took at least 50 min for twitch tension to
return to control levels. After a 50-min wash of the fibers with
drug-free Ringer's solution, twitch tension was 12 ± 8%
(n = 3) of that recorded prior to exposure of the fibers to LASSBio-294 (25 µM).
Effects of LASSBio-294 on Time Course of Twitch Tension.
The
time course of development of twitch tension was only slightly altered
by LASSBio-294. The total duration of the twitch was not significantly
altered after 40-min perfusion of the skeletal muscle fibers with
Ringer's solution containing LASSBio-294 (25 µM). Time to peak
tension (Tp) and relaxation times at 50%
(T0.5) and 80%
(T0.8) of peak force were about the same in
the absence and in the presence of the drug (Table
1). The maximal rate of tension
development (+TVmax) during the twitch was
also not altered by LASSBio-294 (Table
2). Although LASSBio-294 (25 µM) had no significant effect on the total duration of the twitch, it accelerated the maximal rate of tension decay
(
TVmax).
TVmax was approximately 1.18 times faster
in the presence than in the absence of the drug (Table 2).
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TABLE 1
Time course of twitch tension development in single phasic skeletal
muscle fibers in the absence and in the presence of LASSBio-294
Values were estimated from recordings obtained before and 40 min after
perfusion of single skeletal muscle fibers with Ringer's solution
containing LASSBio-294 (25 µM). Results are presented as mean ± S.E.M. (n = 3). According to the unpaired Student's
t test, all results obtained from muscle fibers under
control conditions were not significantly different from those obtained
from muscle fibers exposed for 40 min to LASSBio-294.
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TABLE 2
Effects of LASSBio-294 on the maximal rates of twitch tension
development and decay
Ratios express the maximal rate values estimated from recordings
obtained 40 min after perfusion of single skeletal muscle fibers with
Ringer's solution containing LASSBio-294 (25 µM) divided by the
values estimated from recordings obtained under control condition.
Results are presented as mean ± S.E.M. (n = 3).
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LASSBio-294 Causes No Changes in Resting Membrane Potential or
Action Potentials of Frog Sartorius Muscle.
The positive inotropic
effect of LASSBio-294 could be explained by changes in the first step
of the e-c coupling, i.e., the sarcolemmal action potential. If
LASSBio-294 would, for example, prolong the action potential and,
thereby the mechanically effective period, it could increase the amount
of the contractile activator Ca2+ released. The
effect could also be explained by sarcolemmal
Ca2+ conductance changes that would be reflected
in the shape of the action potential and/or in the value of the resting
membrane potential. Therefore, the effects of LASSBio-294 on action and
resting potentials were investigated.
Resting membrane potential of sartorius muscle continuously perfused
with Ringer's solution was about 86 mV and was not altered after
perfusion of the fibers for 30 to 50 min with solution containing LASSBio-294 (12.5 and 25 µM). Also unaltered were the amplitude and
the duration of the action potential (Table
3). Thus, the positive inotropic effect
of LASSBio-294 cannot be accounted for by changes in the conductance
properties of the muscle fibers.
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TABLE 3
Resting and action potentials of frog sartorius muscles are unaffected
by LASSBio-294
Values were estimated from recordings obtained before and 30 to 50 min
after perfusion of frog sartorius muscles with Ringer's solution
containing LASSBio-294 (12.5 or 25 µM). Results are presented as
mean ± S.E.M. (n = 3 fibers from 2 intact
muscles). According to the paired Student's t test, all
results obtained from muscle fibers under control conditions were not
significantly different from those obtained after exposure of the
muscles to LASSBio-294.
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Effects of LASSBio-294 on Fatigue Development.
Muscle fatigue
can be induced by prolonged, direct electrical stimulation that leads
to a state during which the contractile force declines to the point
where the muscle becomes mechanically refractory to further
stimulation. Fatigue is the result solely of contractile failure of the
muscle involved (Garcia et al., 1991). The finding that LASSBio-294 has
a positive inotropic effect in phasic skeletal muscle cells raised the
question of whether the drug could also alter fatigue development. To
answer this question, single fibers of the semitendinous or tibialis
anterior muscles were fatigued by repetitive cyclic tetanic stimuli
(see Materials and Methods) in the presence and in the
absence of LASSBio-294 (12.5, 25, or 50 µM). Recovery after fatigue
development, produced with intermittent repetitive tetanic stimulations
of the type used here, is not always 100%, and the time it takes for
recovery varies from fiber to fiber. Therefore, each fatigue experiment in this series was performed on a different fiber.
LASSBio-294 prolonged the time needed for tetanic force to start
declining (Fig. 4). To compare curves of
fatigue development from different preparations, a fatigue index was
estimated at various times of stimulation by taking the ratio of
maximum tetanic tension produced during every third tetanus to the
tension output in the first tetanus, i.e.,
Tn/To
(Perreault et al., 1993). The relationship between fatigue index
and different cycles of stimulation obtained from several experiments
was significantly altered by LASSBio-294 in a concentration-dependent
manner (Fig. 5). In agreement with
previous studies (Perreault et al., 1993), the rate at which maximal
tetanic tension decayed during development of fatigue changed
continuously, becoming slower as fatigue developed in muscles perfused
with DMSO-containing Ringer's solution (Fig. 5). The fatigue indexes
decreased more slowly at all times in muscle fibers bathed in
LASSBio-294 than in control muscle fibers; the higher the concentration
of LASSBio-294, the slower the fatigue index decayed.
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Fig. 4.
Effects of LASSBio-294 on skeletal muscle fatigue
induced by repetitive tetanic stimulation. Fatigue was induced by
stimulating the single skeletal muscle fibers with 0.8-s tetanic
stimuli repeated every 4.75 s. A single tetanic stimulus was
applied 2.2 s after the end of each tetanic stimulation. The
tetanic tension recorded before the muscle fibers were subjected to the
repetitive stimulation is shown on the left of each panel. Sample
recordings are representative of results obtained from fibers under
control conditions and after 40-min perfusion with Ringer's solution
containing LASSBio-294 (12.5, 25, or 50 µM). The force calibration
bar shown in the second panel from the top applies to the third and
fourth panels.
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Fig. 5.
Effects of LASSBio-294 on time course of fatigue
development in skeletal muscle fibers subjected to repetitive tetanic
stimulation. Fatigue was induced as described in the legend of Fig. 4.
Graph shows plots of fatigue index versus stimulation number and time.
Fatigue index represents the ratio between the tension developed at the
corresponding n, number of tetanic stimulus and the
control prefatigue maximal tetanic force. FTp represents
the time from beginning of the repetitive stimulation to beginning of
fatigue development. FT0.5 and FT0.8 represent
the times from beginning of repetitive stimulation to 50 and 80%,
respectively, reduction of the prefatigue maximal tetanic force.
Symbols and bars represent mean and S.E.M., respectively, of results
obtained from 8 to 10 experiments.
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The effects of LASSBio-294 were also analyzed in three distinct time
segments of fatigue development: FTp,
FT0.5, and FT0.8. Segment
FTp indicates the time from the beginning of the
repetitive stimulation to the beginning of the decrease in fatigue
index, i.e., the beginning of fatigue development. Segments
FT0.5 and FT0.8 correspond
to the time required to decrease the fatigue index by 50 and 80%,
respectively. Table 4 summarizes the
results obtained from all the experiments done with fibers perfused
with DMSO-containing Ringer's solution (control) and with Ringer's solution containing different concentrations of LASSBio-294. All three
segments of fatigue development were prolonged by LASSBio-294 (Table
4). FTp was 52 and 94% longer in the presence of
12.5 and 50 µM LASSBio-294, respectively, than in the absence of the drug. In addition, FT0.5 and
FT0.8 were 41 ± 7.3 and 32 ± 9.4% longer in the presence of 12.5 µM LASSBio-294. Likewise, in the presence of 50 µM LASSBio-294, FT0.5 and
FT0.8 were on average 149 and 282%,
respectively, longer than in the absence of the drug. In other words,
50 µM LASSBio-294 prolonged by approximately 2.5-fold the time needed
for tetanic force to decrease by 80% of the prefatigue tetanic force.
The drug prolonged the time for fatigue to develop, because it
prolonged FTp and slowed down the rate at which
tension declined.
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TABLE 4
Summary of time parameters of fatigue development in the absence and in
the presence of LASSBio-294
Values were estimated from recordings obtained before and 40 min after
perfusion of single skeletal muscle fibers with Ringer's solution
containing LASSBio-294. Results are presented as mean ± S.E.M.
Ratios express the value of each parameter estimated from results
obtained after 40-min perfusion of the fibers with
LASSBio-294-containing physiological solution divided by the respective
parameter estimated from results obtained under control conditions.
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As seen in Table 5, the average slope of
maximal tetanic tension decline during fatigue development from
preparations bathed with 12.5, 25, and 50 µM LASSBio-294 were 1.22, 1.40, and 1.5 times smaller, respectively, than the corresponding
control values. From the average slopes it was estimated that 12, 25, and 50 µM LASSBio-294 would have incremented by 12, 61, and 208%,
respectively, the time needed for maximal tetanic force to reach zero
as a consequence of fatigue development.
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TABLE 5
Effects of LASSBio-294 on fractional tension decay and on estimated
time to reach zero force during fatigue development
Values were estimated from recordings obtained before and 40 min after
perfusion of single skeletal muscle fibers with Ringer's solution
containing LASSBio-294. Results are presented as mean ± S.E.M.
(n = 3 for each experimental condition). Ratios express
the value of each parameter estimated from results obtained after
40-min perfusion of the fibers with LASSBio-294-containing
physiological solution divided by the respective parameter estimated
from results obtained under control conditions.
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After the muscle cells were fatigued to 50% of the maximal control
tetanic force, the time needed for this tetanic tension to recover to
prefatigue tetanic levels was significantly decreased by
LASSBio-294. After tetanic force had decreased to 50% of the prefatigue values during fatigue development, it took an average of 140 min for tetanic force to recover to 80% of prefatigue tetanic force
when the fibers were bathed only with Ringer's solution (Fig.
6). In contrast, in fibers that were
continuously perfused with 25 and 50 µM LASSBio-294, it took on
average 40 and 25 min, respectively, for tetanic force to recover to
nearly 80% of prefatigue values (Fig. 6).
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Fig. 6.
Effect of LASSBio-294 on recovery time after fatigue
development. Plot of the time it takes for 80% prefatigue tension
recovery after fatigue developed to 50% of the maximal pretetanic
force under control conditions and after 40-min perfusion of the fibers
with Ringer's solution containing LASSBio-294 (25 or 50 µM). Fatigue
was induced as described in the legend of Fig. 4. Each graph and error
bar represent mean and S.E.M., respectively, of results obtained from
three experiments. *p < 0.05, according to the
unpaired Student's t test.
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Discussion |
This study demonstrates that the novel thienylhydrazone
LASSBio-294 has positive inotropic effects in single fibers of frog phasic skeletal muscles. It also shows that in skeletal muscles the
compound reduces development of fatigue and accelerates the recovery of
maximal tetanic tension after fatigue is developed. LASSBio-294 can,
therefore, be effective to treat skeletal muscle fatigue associated
with e-c coupling dysfunctions resulting from decreased cyclic AMP
content and/or responsiveness of skeletal myocytes.
LASSBio-294 Increases Force of Contraction of Single Skeletal
Muscle Fibers by Altering SR Ca2+ Uptake and Release.
The positive inotropic effect of LASSBio-294 was characterized by an
increase in twitch tension, an acceleration of the maximal rate of
tension decay during relaxation, and no significant changes in the
maximal rate of tension development or twitch duration. In addition, it
was inversely proportional to the frequency of stimulation, becoming
negligible at 60 and 90 Hz. At these frequencies, Ca2+ released from the SR maintains cytosolic
Ca2+ levels above the binding capacity of
troponin C. Thus, maximal tetanic force induced by stimuli of such
frequencies can only be potentiated by a compound that increases
phosphorylation of the actomyosin complex, which modulates the
Ca2+ sensitivity of the actomyosin-ATPase
complex. The finding that LASSBio-294 does not increase maximal tetanic
force at 60 or 90 Hz strongly suggests that this novel thienylhydrazone
acts by increasing cellular Ca2+ levels.
The positive inotropic effect of LASSBio-294 cannot be attributed to
changes in Ca2+ entry into the muscle cell
(Curtis, 1970). First, if LASSBio-294 altered
Ca2+ conductance, it would have affected the
action potential and/or the resting membrane potential of the single
muscle fibers. Second, changes in Ca2+
conductance appear to have no important role in e-c coupling or twitch
development in phasic skeletal muscles (Gonzalez-Serratos et al.,
1982).
Increased activity of the SR Ca2+ ATPase is the
mechanism that best explains the inotropic effect of LASSBio-294. The
increased activity of the pump results not only in larger SR
Ca2+ accumulation but also in larger efficiency
of clearance of cytosolic Ca2+. The extra
Ca2+ accumulated in the SR, once released during
activation, increases twitch tension. On the other hand, faster removal
of the extra Ca2+ released during activation
results in decreased binding of Ca2+ to troponin
C, thus accelerating the relaxation process. The bell-shaped
relationship between LASSBio-294 concentration and twitch tension is
likely the result of the effects of increased SR
Ca2+ uptake partially counteracting those of
increased SR Ca2+ release.
The slow onset and development of the effect of LASSBio-294 on twitch
tension are in agreement with an intracellular mechanism of action. The
slow reversibility of the effect of LASSBio-294 on twitch tension can
be explained by 1) the high lipophilicity of the compound, 2) the time
it takes for the increased SR Ca2+ content to
return to control levels, and/or 3) slow recovery of metabolic changes
that might underlie the effect. Potentiation of twitch tension by
substances that increase SR Ca2+ content by
interfering with the cyclic AMP metabolism in frog skeletal muscles is
also slowly reversible (Kirchberger et al., 1974; Gonzales-Serratos et
al., 1981).
LASSBio-294 Decreases Muscle Fatigue Development: Involvement of
Cyclic AMP-Dependent Mechanisms.
LASSBio-294 reduced fatigue
development of skeletal muscle fibers and accelerated recovery of
maximal tetanic tension after fatigue developed. There are two main
theories regarding the cellular mechanisms of fatigue. One theory,
referred to as the metabolic hypothesis, suggests that fatigue of type
II (fast-twitch) skeletal muscles is caused by alterations in the
intracellular concentrations of ATP hydrolysis by-products
(Pi, H+, and
Mg2+) that result in decreased force-generating
capacity (Godt and Nosek, 1989; Chin and Allen, 1998). According to the
other theory, referred to as the e-c coupling hypothesis, during
repetitive stimulation, there is substantial K+
accumulation in the transverse tubular system. The
Na+-K+ ATPase, which is
localized in the T-system (Dombrowski et al., 1996), then operates at
full capacity and becomes unable to remove the excess of tubular
K+ (Clausen, 1996). The increased tubular
K+ concentration depolarizes the tubular
membrane, causing failure in generation and/or propagation of tubular
action potentials and reduction of SR Ca2+
release. Fatigued muscles also have a swollen T-system
(Gonzalez-Serratos et al., 1978; Somlyo et al., 1978) that
contributes to uneven conduction of action potentials along the
T-system (Juel, 1988) and/or to improper signaling between the T-system
and the terminal cisternae of the SR (Garcia et al., 1991). Reduced SR
Ca2+ release in fatigued muscles, which is
accompanied by increased resting levels of cytoplasmic
Ca2+ (Chin and Allen, 1996) and myofibril
inactivation (Garcia et al., 1991; Edman and Lou, 1992), especially
during the fast decay of tension development, appears to be a
predominant factor in the progression of fatigue. Fatigue development
induced by repetitive tetanic stimulations of the type used here
involves both metabolic and e-c coupling mechanisms.
Alleviation of fatigue development takes place when cytosolic levels of
cyclic AMP are increased in type I, slow-twitch (Juel, 1988; Chen and
Alway, 2001) and type II, fast-twitch (Clausen, 1996) skeletal muscle
fibers as well as in the diaphragm, which has both types of fibers
(Kolbeck and Speir, 1991). Thus, the effects of LASSBio-294 on muscle
fatigue can be explained by several mechanisms that have a common start
point, i.e., cyclic AMP. These mechanisms include, but are not
restricted to 1) cyclic AMP-dependent phosphorylation of ryanodine
receptors (Cairns et al., 1993); 2) indirect activation of SR
Ca2+ ATPase by cyclic AMP-dependent
phosphorylation of phospholamban (Kirchberger et al., 1974;
Gonzalez-Serratos et al., 1981; Schwinger et al., 1999); and 3) cyclic
AMP-dependent stimulation of the Na+-K+ ATPase, which
reduces tubular K+ accumulation allowing full
generation and propagation of tubular action potentials.
PDEs as Potential Molecular Targets for LASSBio-294.
An
increase of cellular cyclic AMP levels by LASSBio-294 could explain the
effects of the drug on muscle twitch and fatigability. The pleiotropic
nature of the intracellular effects of cyclic AMP makes this an
attractive explanation of the drug's effect, because only the
simultaneous interaction with several of the relevant targets could
have explained the positive inotropic and antifatigue effects of
LASSBio-294.
There are two possible mechanisms by which LASSBio-294 could increase
cytosolic cyclic AMP concentration. One encompasses direct stimulation
of cyclic AMP production, possibly mediated by the interaction of
LASSBio-294 with adenylate cyclase, Gs proteins, or 
-adrenoceptors (Gonzalez-Serratos et al., 1981; Van Der Heijden et al., 1998). Another consists of PDE inhibition, and consequently, reduction of cyclic AMP breakdown. Inhibition of different PDE isoforms
has positive inotropic effects in cardiac and skeletal muscles
(Gonzalez-Serratos et al., 1982; Alvarez et al., 1986; Morner
and Mansson, 1990; Bishop et al., 1998). In addition to the cyclic
AMP-specific PDE4, which is bound to myofibrils and is present in the
SR of skeletal muscles (Worby et al., 1992; Francis et al., 2001, and
references therein), several PDE isoforms have been found in skeletal
muscles, including PDE1B, PDE2, PDE3, PDE5A, PDE7B, and the newly
characterized PDE11 (Ball et al., 1980; Löbbert et al., 1996;
Francis et al., 2001, and references therein).
Although the exact molecular target for LASSBio-294 is yet to be
identified, it is tempting to speculate that the effects of the drug
are mediated by its interaction with a PDE isoform. First, the effects
of LASSBio-294 in frog skeletal muscles resemble those of other PDE
inhibitors (Mansson and Edman, 1985; Morner and Mansson, 1990). Second,
this thienylhydrazone is a bioisoster of pyridazinone compounds that
are selective and potent inhibitors of PDE4 (Piaz et al., 1997). Third,
LASSBio-294 has cardiotonic properties that can be explained by PDE
inhibition in the cardiovascular system (Sudo et al., 1998).
Potential Therapeutic Applications of LASSBio-294.
Abnormal
skeletal muscle contractility, metabolism, and easy fatigability are
major debilitating symptoms in patients with CHF (Buller et al., 1991).
Phasic skeletal muscles from rats with CHF also have abnormalities in
e-c coupling and fatigue faster than muscles from normal rats
(Perreault et al., 1993). These changes are very similar to those seen
in the myocardium of laboratory animals and humans with CHF (Morgan,
1991) and are most likely due to reduced cyclic AMP content and/or
responsiveness of the skeletal muscle fibers without atrophy (Perreault
et al., 1993; Bishop et al., 1998). Indeed, increasing cyclic AMP
levels not only ameliorates cardiac function but also alleviates the
depressed twitch tension and reduces fatigue of skeletal muscles in
animal models of CHF (Grossman et al., 1996; Bishop et al.,
1998). Thus, it is conceivable that, by increasing cyclic AMP,
LASSBio-294 can improve skeletal muscle function in CHF patients.
Exercise tolerance is an important predictor of survival and quality of
life in CHF patients. In fact, many investigators believe that
therapeutic interventions designed to improve skeletal muscle function
in CHF patients are superior to those designed exclusively to increase
cardiac muscle inotropism (Blackwood et al., 1990, and references
therein). Therefore, LASSBio-294, by virtue of its ability to increase
inotropism and reduce fatigue of skeletal muscles, is a potential
candidate compound for treatment of CHF.
This work was supported in part by funds from the Programa de
Núcleos de Excelência (PRONEX) 0888/96 and the Conselho
Nacional de Desenvolvimento Científico e Tecnológico
(CNPq). The compound LASSBio-294 is currently filed under the Patent
Cooperation Treaty application number 17024.
CHF, chronic heart failure;
e-c, excitation-contraction;
PDE, phosphodiesterase;
DMSO, dimethyl
sulfoxide;
FT, fatigue tetanic tension;
SR, sarcoplasmic reticulum;
T, tetanic tension.
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Abstract
Introduction
a gene involved in fat metabolism located at 11p 15.1.
Genomics
37:
211-218[Medline].
