Cardiac-like behavior of an insect flight muscle
Department of Biology, University of Washington, Seattle, WA 98195-1800, USA
* Author for correspondence (e-mail: mstu{at}u.washington.edu)
Accepted 20 April 2004
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Summary |
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Key words: flight, strain, Manduca sexta, lengthtension, muscle
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Introduction |
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For flying animals, in vivo length changes of muscles have been
measured for birds (Biewener et al.,
1998; Dial and Biewener,
1993
; Williamson et al.,
2001
) and for asynchronous flight muscles in insects (bumblebees
Gilmour and Ellington,
1993
; Josephson and Ellington,
1997
; Drosophila
Chan and Dickinson, 1996
).
These studies, however, do not locate the in vivo operating length
range with respect to each muscle's lengthtension curve. More data are
available for estimates of in vivo sarcomere length ranges during
terrestrial locomotion and swimming (reviewed by
Burkholder and Lieber, 2001
).
However, these measurements are all referenced to the skeletal muscle
lengthtension curve measured for frog leg muscles by Gordon et al.
(1966
). Two issues potentially
confound previous analyses. First, the shape, and particularly the steepness,
of the lengthtension curve depends strongly on the level of muscle
activation (Rack and Westbury,
1969
). Because few muscles are maximally activated during
locomotion, estimates of the in vivo operating range of a muscle
referenced to the lengthtension curve for tetanically activated muscles
may not accurately reflect in vivo performance. Second, variation in
filament geometry cannot account for all of the known variation in the
lengthtension relationship, in particular the well-known differences
between curves for cardiac and skeletal muscles
(Allen and Kentish, 1985
;
Layland et al., 1995
).
Despite the importance of the operating length range, there are surprisingly few studies in which the in vivo length changes of a locomotor muscle have been mapped onto its own lengthtension relationship measured at levels of activation similar to those that occur during locomotion. Such measurements are technically challenging, and many muscles, such as those in arms, fins and legs, generate and control motions that are highly variable and complex. The resulting uncertainty in operating length complicates analyses of structural adaptations and performance. On the other hand, muscles that are restricted to repetitive length changes at relatively constant frequency and amplitude may represent simpler cases that may more readily yield insights into muscle design.
Cardiac muscle is one case where the functional consequences of operating
length are well known. Three emergent properties are critical for cardiac
muscle performance: (1) cardiac muscles operate at strains as high as 10%
(Layland et al., 1995); (2)
such strains occur at sarcomere lengths that lie entirely on the ascending
portion of the cardiac lengthtension relationship
(Layland et al., 1995
) and (3)
compared with skeletal muscle, cardiac muscle shows an exceptionally steep
lengthtension relationship that cannot be accounted for solely by the
changes in myofilament overlap (Allen and
Kentish, 1985
; Layland et al.,
1995
). In addition to geometric components, regulatory factors
such as the particular troponin isoform can affect the steepness of the
forcelength relationship (Gordon et
al., 2000
). Regardless of the underlying mechanisms, however, the
steep lengthtension relationship clearly plays a crucial role in
cardiac muscle performance. Regulation of cardiac output by changes in
end-diastolic volume, as described by the FrankStarling principle,
requires greater muscle forces to accommodate greater ventricular filling.
Operation at lengths that lie on the ascending portion of a steeply rising
lengthtension curve gives cardiac muscle the capacity to generate these
larger stresses as the ventricle walls stretch
(Allen and Kentish, 1985
;
Layland et al., 1995
). The
possibility that skeletal muscles might also exploit these design principles
has not been explored. Because the muscles that power insect flight also
undergo relatively constant, cyclic length oscillations, we were intrigued by
the possibility that insect flight muscle and mammalian cardiac muscle may
share fundamental mechanical characteristics.
Insects have evolved two distinct types of flight muscle. One type,
asynchronous muscle, powers flight in many small insects (e.g. Diptera,
Hymenoptera). Here, decoupling between the timing of neuronal activation and
muscle contraction permits high-frequency contractions without the need for
elaborate internal membrane systems for calcium cycling
(Josephson et al., 2000).
Asynchronous flight muscle is a unique and critical adaptation that permits
high power output at high contraction frequencies, within the stringent weight
and volume constraints imposed on small flying insects. Many insects, however,
power flight with synchronous muscle. In these muscles, as in the flight
muscles of bats and birds, muscle contraction is directly coupled to motor
axon activation. Insects with synchronous flight muscle therefore offer an
opportunity to draw parallels between insect and vertebrate fliers as we seek
to understand general design principles for animal flight. Central to this
effort are detailed analyses of the patterns of force production and
strain.
Here, for a synchronous insect flight muscle, the mesothoracic
dorsolongitudinal muscles (dl1 muscles;
Nüesch, 1953;
Eaton, 1988
) of the hawkmoth
Manduca sexta, we report both in vivo muscle length changes
and the length dependence of isometric twitch forces measured using a
physiologically appropriate level of stimulation. We use these data to
determine the in vivo operating length range of the dl1
muscles relative to their active twitch lengthtension relationship. In
a subsequent paper (M.S.T. and T.L.D., in preparation), we examine the effects
of mean operating length, strain amplitude and phase of activation on the
mechanical power output of the dl1 muscles.
The dl1 muscles of Manduca provide an ideal system for
addressing questions of muscle design. These synchronous muscles power the
downstroke of the wings. Like cardiac muscles, the dl1 muscles
drive highly repetitive motions. Alterations in wing stroke kinematics appear
to be accomplished by a separate, anatomically distinct set of steering
muscles (Kammer, 1985). The
ability to reliably define a reference length is critical for any analysis of
length effects on muscle function. Fortunately, while at rest,
Manduca assume a stereotyped posture with their wings folded back
over their body. This behavior allowed us to unambiguously identify a
consistent anatomical rest length, Lr, of the
dl1 muscles. In this study, we measure length changes of the
dl1 muscles in tethered flight and map these length changes onto
their isometric twitch lengthtension curve. Because the dl1
muscles are typically activated once in each wing stroke
(Kammer, 1971
;
Rheuben and Kammer, 1987
;
Wendler et al., 1993
), we
measure the isometric twitch lengthtension curve rather than a
lengthtension curve based on tetanic force.
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Materials and methods |
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The dl1 muscles span the length of the mesothorax and attach to
the 1st and 2nd phragmata (Fig.
1). The phragmata are deep invaginations of the dorsal exoskeleton
that form broad areas for muscle attachment. Each of the bilaterally paired
dl1 muscles consists of five sub-units designated, from ventral to
dorsal, dl1a to dl1e
(Nüesch, 1953;
Eaton, 1988
).
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Experimental approach
Due to anatomical constraints and the conflicting characteristics of the
instrumentation required to measure in vivo length changes and
isometric force, we could not perform all measurements on the same individual
moths. We therefore used a two-pronged approach. First, during tethered
flight, we measured the amplitude of muscle length changes and the mean
operational length, both referenced to the anatomical rest length
(Fig. 1). Second, in a group of
intact moths, we measured the active component of the isometric twitch force
to generate the isometric twitch lengthtension curve for the
dl1 muscles (Fig.
1). As with the measurements of length changes during tethered
flight, these lengthtension measurements were also referenced to the
anatomical rest length. Using the anatomical rest length as a reference for
both sets of measurements enabled us to map in vivo length changes
during flight onto the isometric twitch lengthtension curve.
Preservation of this reference length in our active twitch lengthtension measurements required that we leave the exoskeleton intact. Because we measured only the active component of muscle twitch force, our measurements were not contaminated by the steady-state stress in the exoskeleton due to imposed changes in muscle length. To assess the extent to which the intact exoskeleton may have altered the shape of the measured twitch lengthtension curve, we performed a second set of twitch lengthtension measurements on mechanically isolated muscles (Fig. 2). The measurements on mechanically isolated muscles also permitted force measurements over a larger range of length than was possible in the intact thorax.
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Muscle strain and activation phase during tethered flight
Moth preparation
We prepared moths under normal room light near the end of the light phase
of their photoperiod. Moths were immobilized by 12 min of exposure to
CO2. We rubbed off the scales of the meso- and metathoracic coxae
and glued a tapered brass rod between the coxae with cyanoacrylate adhesive.
The rod was then clamped with the longitudinal body axis of the moth oriented
horizontally. Tethered flight data were recorded under low light conditions
during the first 13 h of the moth's normal period of darkness,
23 h following CO2 exposure.
Length transducers
We measured length changes of the dl1 muscles by tracking the
motions of the 1st and 2nd phragmata simultaneously. We used two displacement
transducers, each consisting of a spring with a probe attached to its free end
(Fig. 1). The probes were
constructed from 0.78 mm-diameter stainless steel hypodermic tubing. Each
ended in a small, upturned hook. We used optical sensors (Spot 2D; UDT Sensors
Inc., Hawthorne, CA, USA) paired with red light-emitting diodes (LEDs) to
track the motion of a small metal flag soldered to the base of each probe. The
fixed end of each probe, together with its associated sensor, was mounted on a
linear translation stage. To calibrate each transducer, the hook was clamped
to a fixed reference, and the voltage output of the optical sensor amplifier
circuit was recorded as the translation stage was moved in known increments.
The micrometer of the translation stage allowed us to calibrate each
displacement transducer to a precision of 10 µm.
The anterior probe was hooked onto the ventral margin of the 1st phragma through a short, transverse incision in the membranous region just anterior to the metathoracic prescutum. We inserted the probe of the posterior displacement transducer through a narrow slit cut in the soft cuticle along the dorsal midline of the first two abdominal segments. This probe deflected the heart to one side, passed through a large abdominal air sac, and hooked onto the ventral margin of the 2nd phragma. Coupled motion of the wings and probes indicated successful attachment of the probes.
The anterior and posterior displacement transducers had unloaded resonant frequencies of 50 and 40 Hz, respectively, well above the typical wing-beat frequency of approximately 25 Hz. Both transducers had a compliance of 0.01 m N-1. Based on subsequent measurements of twitch forces, tension imposed on the dl1 muscles by the displacement transducers was maximally 6% of the peak isometric twitch force when the muscle was stretched by 10% of its resting length.
To maintain stable contact with the phragmata, we positioned each transducer so that its spring was under light tension. This tension was not high enough, however, to alter the rest position of the wings. To further verify that the tension in the displacement transducers did not change the rest length of the muscles, we performed dissections on several moths to expose the ventral surface of the dl1 muscles, with the displacement transducers attached. Stretching the springs of the displacement transducers to lengths beyond those observed in flight did not produce visible movements of the phragmata, indicating that the tension in the transducers did not bias our measurements of muscle length.
EMG recordings
We performed differential EMGs from the left dl1c because it is
the largest sub-unit that is directly accessible through the scutum. We used
pairs of electrodes fashioned from short lengths of 25.4 µm
formvar-insulated Nichrome wire (38.1 µm outside diameter; A-M Systems,
Carlsborg, WA, USA), with the insulation cut flush with the end of the wire.
After removing the scales covering the anterior insertion of dl1c,
we implanted both electrodes through holes in the cuticle made by a minuten
pin. Extracellular potentials were amplified using a differential AC amplifier
(Model 1700; A-M Systems) and band pass filtered (0.320 kHz).
Data acquisition and analysis
We recorded extracellular potentials and muscle length changes using a
16-bit analog-to-digital converter interfaced with a computer. Each data
channel was digitized at a rate of 4 kHz. We simultaneously recorded all
trials on video tape at 30 frames s-1 using a digital video camera.
The video camera was oriented perpendicular to the sagittal plane of the moth.
A mirror placed at a 45° angle in front of the moth provided a view of the
wing stroke in the transverse plane. An LED placed in the video field was
illuminated during data acquisition and allowed us to synchronize the computer
and video recordings.
We define the anatomical rest length, Lr, as the length of dl1a along its ventral surface in the intact thorax of a quiescent moth. We measured Lr in tethered flight preparations as the distance separating the hooks of the displacement transducer probes (Fig. 1). At the end of a recording session, we fixed the distance between the hooks by gluing two strips of acetate transparency film across the space separating the two probes. The acetate strips held the probes in position while we disengaged the probes from the phragmata and removed the moth from the apparatus. We then measured Lr to the nearest 0.05 mm.
For each sample period in the muscle length recordings, we calculated the instantaneous length of the muscle as the sum of Lr and the combined displacements of the 1st and 2nd phragmata away from their rest positions. Each strain cycle was defined as the time separating successive minima in the strain record. Wing-beat frequency was calculated for each cycle and averaged across the total number of complete cycles in each flight sequence. We defined the operational muscle length, Lop, for each cycle as the midpoint between the maximum (Lup) and minimum (Ldn) length of the muscle in that cycle: Lop=(Lup+Ldn)/2. We calculated strain amplitude as the total fractional change in muscle length in each cycle, normalized to the operational length: strain=(LupLdn)/Lop. We then averaged values of strain amplitude and Lop across all wing-strokes of each flight bout. We calculated the phase of activation of the dl1 muscles as the time from the start of muscle lengthening to the peak of the spike in the subsequent extracellular spike, divided by the cycle period.
Isometric twitch and twitch lengthtension measurements
Force transducer
The force transducer consisted of a cantilevered 6.25x1.5 mm brass
beam with a free length of 35.25 mm. We used an optical sensor (Spot 2D; UDT
Sensors Inc.) to track the position of a short length of stainless steel
hypodermic tubing soldered to the end of the beam. The force beam had a
compliance of 3.6x10-4 m N-1 and an unloaded
resonant frequency of 640 Hz. The force transducer was mounted on a 3-axis
micromanipulator, which in turn was mounted on a linear translation stage.
Using the calibrated micrometer on the translation stage, we could adjust the
force transducer position with a precision of 0.01 mm.
Intact thorax preparation
To measure the isometric twitch lengthtension curve in intact moths,
we prepared moths as described for tethered flight experiments. As in the
tethered flight preparation, the phragmata were secured to hooks inserted
through dorsal incisions in the prothorax and abdomen. The hooks differed from
those used for strain measurements in that they were bilaterally paired to
avoid slipping or tearing the cuticle as the muscles twitched. Instead of
using compliant displacement transducers, the probe hooked onto the anterior
phragma was rigidly fixed in place, and the posterior probe was connected
directly to the force transducer.
Any disruption of the thoracic exoskeleton invariably alters the resting length of the muscles. Consequently, with the exception of the incisions in soft tissue of the prothorax and abdomen required to insert the displacement transducer probes, we left the thoracic exoskeleton completely intact. We also monitored wing posture to detect any changes in the relative positions of the phragmata that occurred when we attached the hooks. We removed any length offset introduced during hook placement by adjusting the hook position until the wings assumed their normal resting posture.
Isolated muscle preparation
The dl1 muscles receive their primary respiratory air supply
from large tracheal trunks originating at the mesothoracic spiracles. On each
side of the moth, these tracheal trunks run anteriorly between the
dl1 muscles and the dorsoventral muscles, supplying both muscle
groups. This arrangement of tracheae made it impossible to remove the
dl1 muscles from the thorax without compromising their oxygen
supply. In addition, respiratory pumping by the abdomen appears critical for
prolonged viability of the dl1 muscles; muscle performance
deteriorated rapidly if we removed the abdomen in order to expose the
posterior muscle attachments on the 2nd phragma. We therefore developed a
semi-intact preparation that minimized the dissection necessary to
mechanically isolate the dl1 muscles between two specially
constructed grips (Fig. 2).
The anterior grip consisted of a small aluminum block shaped to match the contours of the anterior mesoscutum and the 1st phragma (Fig. 2). Each moth was immobilized by brief (12 min) exposure to CO2, decapitated, and the scales rubbed off from the dorsal surfaces of the thorax. Once the thoracic exoskeleton is cut to mechanically isolate the dl1 muscles, wing position no longer serves as a reliable indicator of rest length. We therefore removed the wings to facilitate the muscle preparation. The 1st phragma was exposed by severing the pronotum near its articulation to the mesothoracic prescutum and by clearing away the prothoracic dorsolongitudinal muscles. After scraping away the waxy epicuticle, we used cyanoacrylate adhesive to attach the grip to the cuticle overlying the anterior origins of the dl1 muscles. Any gaps between the grip and the exoskeleton were filled with a composite formed from cyanoacrylate and sodium bicarbonate powder.
The posterior grip consisted of a pair of 0.68 mm-diameter stainless steel hypodermic needles soldered to a small brass block (Fig. 2). The needles were parallel to each other and were separated by a distance slightly less than the lateral width of the 2nd phragma. A drop of cyanoacrylate adhesive was placed in the deep groove overlying the phragma. We then pushed the needles down into the groove so that they punctured the metathoracic scutellum and passed down along the posterior face of the phragma. Cyanoacrylate flowing between the needles and the cuticle solidly bonded the phragma and scutellum to the needles and brass block. We discarded trials if dissection following the measurements showed that the needles had pierced the phragma, or if the needles and phragma were not solidly bonded.
To preserve the orientation of the dl1 muscles as the thorax was transferred to the experimental apparatus, we glued two strips of acetate transparency film, one strip on each side, across the gap separating the two grips. The acetate strips restricted length changes and prevented bending and torsion of the muscles. We then excised a thin strip of cuticle from around the anterior grip. After cutting away this strip of cuticle, the dl1 muscles and the acetate strips glued to the grips were the only remaining mechanical links between the anterior and posterior attachments of the muscles.
The anterior grip was attached to the force beam via a short, threaded steel rod projecting from the front of the grip. The threaded end of the rod was inserted through a hole near the end of the force beam and secured with a nut. The posterior grip was attached to a linear translation stage. A short length of stainless steel tubing projected from the back of the grip and terminated in a ball bearing. The ball bearing fit into a depression at the end of a threaded rod rigidly fixed to the translation stage. The threaded rod and the ball bearing formed a ball joint when secured with a slotted retaining nut.
Once the two grips were secured, we cut the acetate strips connecting the two grips, leaving the dl1 muscles as the only direct mechanical linkage between the fixed posterior section of the thorax, and the anterior muscle attachments connected to the force beam. Cutting the acetate strips also relieved any stresses induced in the thorax during the mounting procedure, causing small shifts in the relative positions of the two grips. We used the ball joint and the micromanipulator mount of the force beam to restore the initial muscle length and orientation. The position of the muscle was adjusted until both cut edges of both acetate strips were just touching and exactly aligned. The force beam manipulator was then locked in position and the retaining nut of the ball joint was tightened to prevent movement of the posterior grip. Finally, the acetate strips were trimmed away to prevent mechanical interference with imposed muscle length changes.
Temperature measurement and control
We measured thoracic temperature to the nearest 0.1°C using a 0.15
mm-diameter copperconstantan thermocouple inserted into the
dl1 muscles through a small hole in the cuticle. The entire
experimental apparatus was enclosed within an insulated plywood box. Heated
water circulating through copper pipe within the box allowed us to maintain
thorax temperature at 36±0.5°C. All twitch lengthtension
measurements were carried out at 36°C, the minimum thoracic temperature
required for flight (Heinrich and
Bartholomew, 1971; McCrea and
Heath, 1971
).
Muscle stimulation
We elicited twitches using bipolar, supramaximal stimuli, 0.2 ms in
duration, delivered through a pair of stainless steel minuten pins. The
minutens were inserted through the anterior notum and into the dl1
muscles, one on ether side of the midline. In the intact thorax preparation,
spurious stimulation of the antagonistic dorsoventral muscles required a
doubling of the stimulus amplitude, and the resulting forces were clearly
evident as both an upwards deflection of the wings combined with a reversal in
the sign of the recorded twitch force. Stimuli were delivered continuously, at
a frequency of 10 Hz. At this frequency there was no detectable fusion between
successive twitches.
Measurement protocol and analysis: intact thorax and isolated muscles
We used both isolated muscle and intact thorax preparations to examine the
effects of length on active twitch force. Twitch forces were digitized at a
sample rate of 2 kHz. In all cases, we report the active twitch force, the
peak force relative to the baseline for each twitch.
To examine the effects of muscle length on active twitch force, we changed muscle length in steps of 0.1 mm (isolated muscle) or 0.2 mm (intact thorax). We recorded either one or two complete ascending and descending length series from each preparation. In the isolated muscle preparations, we recorded active twitch forces over a range of lengths from 0.84 to 1.15 Lmax. In the intact thorax preparation, the minimum length was Lr since the probes did not allow us to apply compressive forces to the muscle.
At the conclusion of each experiment, we used the calibrated micrometer on
the translation stage to return the dl1 muscles to their initial
length. Two strips of stainless steel shim, one on each side, were glued
across the gap separating the grips, securing the muscle at its initial
length. We then removed the preparation from the experimental apparatus and
dissected away the thorax surrounding the dl1 muscles, leaving the
muscles and their sites of attachment in place between the two grips. Using
digital calipers, we measured the distance between the ventral and medial
margins of the 1st and 2nd phragmata to the nearest 0.01 mm. The
dl1 muscles of one side were dissected free of the thorax and
placed in Manduca saline (Tublitz
and Truman, 1985). We recorded a video image of the medial aspect
of the intact, contralateral dl1 muscles. The remaining
dl1 muscles were then dissected free of the thorax and also placed
in saline. Finally, the muscles were blotted dry and weighed together to the
nearest 0.001 g. We calculated muscle volume from the measured muscle mass,
assuming a muscle density of 1 g cm-3. To calculate mean fiber
length, we used six measurements of fiber length taken at evenly spaced
intervals across the medial surface of the dl1 muscles on the video
image. We calculated the combined cross-sectional area of the two
dl1 muscles from the muscle volume divided by the mean fiber
length.
To generate lengthtension curves from the isolated muscle and intact thorax preparations, we first fit 2nd order polynomial curves (polyfit; Matlab) to the individual twitch lengthtension data sets, then averaged the polynomial coefficients for all individuals within each of the two types of preparations. We determined the length at maximum force, Fmax, from the polynomial fit of the lengthtension data for each individual.
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Results |
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Muscle strain and activation phase in vivo
The moths used in our tethered flight measurements initiated pre-flight
warm-up either spontaneously or in response to gentle prodding with a wooden
applicator stick. Following an initial 510 min warm-up period, the
moths switched to the large-amplitude wing stroke characteristic of normal
flight. All data were collected during sustained flight bouts, at least
1520 min following the initiation of flight behavior.
We selected flight sequences for detailed analysis if the dl1 muscles fired continuously in every wing stroke, wing-beat frequency was 20 Hz or higher, and the video record showed that stroke amplitude was large and relatively constant. From these sequences, we analyzed one representative flight sequence for each of six moths (Table 1). The selected sequences were 59106 wing-beats in duration with wing-beat frequencies of 21.125.6 Hz.
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Length changes of the dl1 muscles were approximately sinusoidal (Fig. 3). To compare the spectral composition of the muscle strain records across individuals, we normalized the Fourier-transformed data to the fundamental frequency. Distortion of the strain waveform, measured as the amplitude of the 2nd harmonic expressed as a percentage of the 1st harmonic, varied from 5.5 to 46%, with a mean of 25±17% (S.D.; N=6; Fig. 4; Table 1).
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During flight, there was a net shortening of the dl1 muscles so that, on average, Lop was 0.98±0.02 Lr (S.D.; N=6). In each wing stroke, the total peak-to-peak amplitude of muscle strain was 0.09±0.02 Lop. In five of the six moths, lengthening lasted slightly longer than shortening. On average, the duration of muscle shortening was 45±5% of the cycle period. The dl1 muscles typically fired just before the onset of muscle shortening, with an average phase, relative to the muscle length change cycle, of 0.49±0.04.
Operating range of the dl1 muscles on their twitch lengthtension curve
The mean ratio of the anatomical rest length to the length at maximum
isometric tension (Lr/Lmax) measured
in intact thorax preparations was 0.91±0.03 (S.D.;
N=7). This ratio and the ratio
Lop/Lr from our in vivo
length experiments enabled us to calculate an estimate of the position of
Lop on the isometric twitch lengthtension curve:
Lop/Lmax=(Lop/Lr)(Lr/Lmax).
Based on this calculation, the mean Lop of the
dl1 muscles was 0.89±0.04 Lmax
(Fig. 5). This
Lop, together with the average peak-to-peak in
vivo strain amplitude of 0.09±0.02 Lop,
indicates that the dl1 muscles operate entirely on the ascending
limb of their twitch lengthtension curve, over a range of
0.850.93Lmax
(Fig. 5). Because the
configuration of the muscle grips used for twitch measurements in the intact
thorax did not permit us to shorten the dl1 muscles, this
operational length range lies almost entirely below the length range of our
isometric twitch measurements. Our results do, however, indicate that within
the operating length range during flight, the maximum isometric twitch force
in the intact thorax was only 0.72Fmax. On the
lengthtension curve measured from mechanically isolated muscles, the
length range of 0.850.93Lmax corresponds to
isometric twitch forces of 0.080.79Fmax.
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The lengthtension curves measured by the two different methods were not identical. The intact thorax preparation produced a somewhat narrower lengthtension curve, and the mean value of peak stress at Lmax was 32±8 kPa (S.D.; N=7), substantially lower than the corresponding value of 82±10 kPa (S.D.; N=4) measured from isolated muscles. These differences, however, do not qualitatively alter our results; in both cases, the Lop range falls entirely on the ascending limb of the lengthtension curve, at muscle lengths where twitch forces were substantially lower than Fmax.
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Discussion |
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To interpret the functional consequences of operation on the ascending limb
of the lengthtension curve, we draw on comparisons with other muscle
systems. Both cardiac muscle and the dl1 muscles undergo cyclic
strains at large amplitudes. The strain amplitude of the dl1
muscles is considerably greater than the largest amplitudes reported to date
for other insects: 3% in the asynchronous wing elevator muscles of
bumblebees (Josephson and Ellington,
1997
). In these asynchronous muscles, molecular mechanisms
associated with stretch activation may restrict the acceptable strains to
rather low amplitudes (Dudley,
2002
). When we compare the twitch lengthtension curves of
the dl1 muscles and mammalian cardiac muscle
(Fig. 6), we see a striking
similarity in shape. Both curves have steep rising regions and narrow peaks.
Moreover, both curves are considerably steeper and narrower than active twitch
lengthtension curves for vertebrate skeletal muscle. The insect
synchronous muscle and the mammalian cardiac muscle also share the functional
characteristic of operating entirely at lengths that fall on the ascending
parts of their lengthtension curves.
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As with mammalian cardiac muscle, the combination of a steep twitch
lengthtension curve and a large operating range on the ascending region
may have important regulatory consequences. Operation on the ascending limb of
a steep lengthtension curve gives mammalian cardiac muscle the capacity
to generate the larger forces required by increases in ventricular filling. In
an analogous manner, operation on the ascending limb of its steep
lengthtension curve may confer the ability of the dl1
muscles to accommodate transient increases in wing stroke amplitude without
the need for changes in neural control. Turning maneuvers by Manduca
involve asymmetrical changes in both wing-stroke amplitude and the extent of
pronation and remotion. These changes appear to be controlled by shifts in the
phase and frequency of activation in the relatively small direct flight
muscles, especially the basalar and axillary muscles
(Kammer, 1971;
Wendler et al., 1993
;
Rheuben and Kammer, 1987
).
Turning maneuvers do not appear to involve substantial changes in the firing
patterns of the indirect dl1 muscles
(Kammer, 1971
). The
dl1 muscles, however, will almost certainly experience transient
changes in strain amplitude as the altered motions of the wings are
transmitted through the mechanical linkages of the thorax. The normally large
amplitude strains in the dl1 muscles, combined with a steep twitch
lengthtension curve, should confer a strong length dependence on force
generation throughout the wing stroke. Moreover, because these length changes
occur entirely on the ascending limb of the twitch lengthtension curve,
the capacity of the dl1 muscles to generate force will rise sharply
as the muscle is stretched. This characteristic of the dl1 muscles
could therefore provide an intrinsic mechanism to restore the amplitude of
muscle length changes to their steady-state value following a transient
increase in strain, even without an adjustment in the pattern of neural
activation. The lowered capacity of the muscles to generate force that results
from operation on the ascending limb of the twitch lengthtension
relationship (vs shortening across the plateau) suggests that such an
intrinsic regulatory mechanism may involve a compromise between control and
power generation. Intrinsic regulation of muscle contraction is generally
thought to be a property restricted to asynchronous flight muscle, in which
stretch activation decouples the amplitude and frequency of muscle
contractions from direct neural control. Our results suggest that synchronous
flight muscles could also have some degree of intrinsic regulation that
depends on their cardiac-like mechanical behavior, even though their
contraction frequency is tightly coupled to the frequency of neuronal
firing.
The range of lengths over which a muscle contracts has a profound influence
on the temporal patterns of its mechanical activity
(Edman and Nilsson, 1971).
Because calcium release occurs more quickly than uptake by the sarcoplasmic
reticulum, twitch force rises much more rapidly than it declines.
For contraction at a constant velocity, this temporal asymmetry will result in greater shortening during the relatively longer falling phase than during the shorter rising phase of a twitch. Therefore, the effect of length dependence on force will be greater during the falling phase. For example, for muscle shortening that occurs entirely down the ascending limb of the lengthtension curve, the decrement in force due to length dependence will be smaller through the short rising phase of a twitch than throughout the longer declining phase.
A muscle that twitches as it shortens along the ascending portion of the lengthtension curve will reach a larger force more rapidly than a muscle contracting along the descending part of the curve. The subsequent decline in twitch force will also occur more rapidly. We illustrate this point using a model of a twitch of a muscle that is allowed to shorten, combined with a simple linear length dependence of the twitch force (Fig. 7).
|
Shortening deactivation may also be important in avoiding fusion in rapidly
contracting cardiac muscles (Allen and
Kentish, 1985). Isometric twitch forces produced by the
dl1 muscles of Manduca show a small degree of fusion when
stimulated at wing-stroke frequency (25 Hz), even in their normal operating
temperature range of 3640°C. Reduced twitch duration due to
shortening deactivation may augment the net power output by reducing active
force generation as the muscle is stretched (negative work).
We do not know the extent to which cardiac-like mechanical behavior may
occur in other skeletal muscle systems. Frog jumping muscles appear to operate
on the plateau of their lengthtension relationship
(Lutz and Rome, 1994). These
muscles, however, power explosive, ballistic motions. Among muscles that power
sustained, cyclic activity, cardiac-like mechanics could provide non-neuronal
mechanisms for regulating the excursion of oscillating legs, wings or other
appendages. Although there are no other additional data for synchronous insect
flight muscles, both a crab flagellar muscle
(Josephson and Stokes, 1987
)
and scallop abductor muscle (Olson and
Marsh, 1993
) operate at lengths shorter than those corresponding
to maximum twitch or tetanic force generation. Intriguingly, the steep tetanic
lengthtension relationship of crab flagellar muscle is similar to those
of mammalian cardiac muscle and the dl1 muscles of Manduca
(Josephson and Stokes, 1987
).
Contrary data for fish, however, suggest that the muscles that power sustained
swimming operate on the plateau region of their tetanic lengthtension
relationship (Rome and Sosnicki,
1991
). Clearly, given the surprising paucity of similar data sets,
generalities may be premature.
We often assume that the shape and slope of the classic
lengthtension relationship published by Gordon et al.
(1966) is a general
characteristic of striated skeletal muscle. Indeed, the conservative range of
sarcomere lengths found among vertebrate striated muscles lends credence to
this assumption. However, a host of data suggests that factors other than
filament geometry alone (and therefore sarcomere lengths) can alter the slope
of the lengthtension relationship: swapping troponins between cardiac
and skeletal muscles, changing the concentration of extracellular calcium
around cardiac cells, and even altering the level of activation for skeletal
muscle, can all change the slope of the lengthtension relationship
(Gordon et al., 2000
). These
findings, taken together with our results, suggest that the mechanical
behavior of locomotor muscles may vary considerably more than previously
thought. Although largely unexplored, such potential variation suggests a
possible range of mechanisms for tuning muscle contractile performance to the
specific functional needs of animal locomotion.
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Acknowledgments |
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References |
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Allen, D. G. and Kentish, J. C. J. (1985). The cellular basis of the length-tension relation in cardiac muscle. J. Mol. Cell. Cardiol. 17,821 -840.[Medline]
Biewener, A. A., Corning, W. R. and Tobalske, B. W.
(1998). In vivo pectoralis muscle force-length behavior
during level flight in pigeons (Columba livia). J. Exp.
Biol. 201,3293
-3307.
Burkholder, T. J. and Lieber, R. L. (2001).
Sarcomere length operating range of vertebrate muscles during movement.
J. Exp. Biol. 204,1529
-1536.
Dial, K. P. and Biewener, A. A. (1993).
Pectoralis muscle force and power output during different modes of flight in
pigeons (Columba livia). J. Exp. Biol.
176, 31-54.
Chan, W. P. and Dickinson, M. H. (1996). In
vivo length oscillations of indirect flight muscles in the fruit fly
Drosophila virilis. J. Exp. Biol.
199,2767
-2774.
Dudley, R. (2002). The Biomechanics of Insect Flight. Princeton: Princeton University Press.
Eaton, J. L. (1988). Lepidopteran Anatomy. New York: Wiley Interscience.
Edman, K. A. and Nilsson, E. (1971). Time course of the active state in relation to muscle length and movement: a comparative study on skeletal muscle and myocardium. Cardiovasc. Res. 1 (Suppl. 1),3 -10.[Medline]
Gilmour, K. M. and Ellington, C. P. (1993).
In vivo muscle length changes in bumblebees and the in vitro
effects on work and power. J. Exp. Biol.
183,101
-113.
Gordon, A. M., Huxley, A. F. and Julian, F. J. (1966). The variation in isometric tension with sarcomere length in vertebrate muscle fibers. J. Physiol. 184,170 -192.[Medline]
Gordon, A. M., Homsher, E. M. and Regnier, M.
(2000). Regulation of contraction in striated muscle.
Physiol. Rev. 80,853
-924.
Heinrich, B. and Bartholomew, G. A. (1971). An analysis of pre-flight warm-up in the sphinx moth Manduca sexta. J. Exp. Biol. 55,223 -239.
Josephson, R. K. and Stokes, D. R. (1987). The contractile properties of a crab respiratory muscle. J. Exp. Biol. 131,265 -287.
Josephson, R. K. and Ellington, C. P. (1997).
Power output from a flight muscle of the bumblebee Bombus terrestris
I. Some features of the dorsoventral flight muscle. J. Exp.
Biol. 200,1215
-1226.
Josephson, R. K., Malamud, J. G. and Stokes, D. R.
(2000). Asynchronous muscle: a primer. J. Exp.
Biol. 203,2713
-2722.
Kammer, A. E. (1971). The motor output during turning flight in a hawkmoth, Manduca sexta. J. Insect Physiol. 17,1073 -1086.[CrossRef]
Kammer, A. E. (1985). Flying. In Comprehensive Insect Physiology, Biochemistry and Pharmacology, vol. 5 (ed. G. A. Kerkut and L. I. Gilbert), pp. 491-552. New York: Pergamon Press.
Layland, J., Young, I. A. and Altringham, J. D. (1995). The length dependence of work production in rat papillary muscles in vitro. J. Exp. Biol. 198,2491 -2499.[Medline]
Lutz, G. J. and Rome, L. C. (1994). Built for jumping: the design of the frog muscular system. Science 263,370 -372.[Medline]
McCrea, M. J. and Heath, J. E. (1971). Dependence of flight on temperature regulation in the moth, Manduca sexta.J. Exp. Biol. 54,415 -435.[Medline]
Nüesch, H. (1953). The morphology of the thorax of Telea Polyphemus (Lepidoptera). I. Skeleton and muscles. J. Morph. Philadelphia 93,589 -608.
Olson, J. M. and Marsh, R. L. (1993).
Contractile properties of the striated adductor muscle in the bay scallop
Argopectten irradians at several temperatures. J. Exp.
Biol. 176,175
-193.
Rack, P. M. H. and Westbury, D. R. (1969). The effects of length and stimulus rate on tension in the isometric cat soleus muscle. J. Physiol. 204,443 -460.[Medline]
Rheuben, M. B. and Kammer, A. E. (1987). Structure and innervation of the third axillary muscle of Manduca relative to its role in turning flight. J. Exp. Biol. 131,373 -402.[Abstract]
Rome, L. C. and Sosnicki, A. A. (1991). Myofilament overlap in swimming carp. II. Sarcomere length changes during swimming. Am. J. Physiol. 260,C289 -C296.[Medline]
Wendler, G. Muller, M. and Dombrowski, U. (1993). The activity of pleurodorsal muscles during flight and at rest in the moth Manduca sexta (L.). J. Comp. Physiol. A 173,65 -75.
Williamson, M. R., Dial, K. P. and Biewener A. A.
(2001). Pectoralis muscle performance during ascending and slow
level flight in mallards (Anas platyrhynchos). J. Exp.
Biol. 204,495
-507.
Tublitz, N. J. and Truman, J. W. (1985). Identification of neurons containing cardioacceleratory peptides (CAPS) in the ventral nerve cord of the tobacco hawkmoth, Manduca-sexta. J. Exp. Biol. 116,395 -410.[Abstract]