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The Journal of Experimental Biology 200, 1473–1482 (1997) Printed in Great Britain © The Company of Biologists Limited 1997 JEB0776
MATURATIONAL CHANGES IN TROPONIN T EXPRESSION, Ca2+-SENSITIVITY AND TWITCH CONTRACTION KINETICS IN DRAGONFLY FLIGHT MUSCLE GAIL H. FITZHUGH* AND JAMES H. MARDEN Department of Biology, Pennsylvania State University, University Park, PA 16802, USA Accepted 12 March 1997 Summary Maximum lift production and the thermal sensitivity of force per unit area than teneral muscle; this difference in lift production increase dramatically during adult force becomes greater at high temperatures. There do not maturation of Libellula pulchella dragonflies. Here, we appear to be any age-related differences in actomyosin report that the mechanistic basis for this transition appears crossbridge properties, since teneral and mature flight to involve a developmental change in protein expression, muscles do not differ in shortening velocity, tetanic tension which alters the Ca2+-sensitivity of muscle activation and or instantaneous power output during isotonic contraction. Thus, variation in TnT expression appears to affect the twitch contraction kinetics. The alternatively spliced Ca2+ temperature-dependent Ca2+-sensitivity regulatory protein troponin T (TnT) undergoes an isoform of muscle shift during adult maturation. Skinned (demembranated) activation, which in turn affects the kinetics and force fibers of mature flight muscle are up to 13 times more production of the twitch contractions used by dragonflies sensitive to activation by Ca2+ than skinned fibers from during flight. This cascade of effects suggests that teneral (newly emerged adult) flight muscle, and their maturational changes in the expression of TnT isoforms Ca2+-sensitivity is more strongly affected by temperature. may be a key determinant of overall muscle and organismal Intact muscle from mature individuals has a shorter time performance. to peak tension and longer time to half-relaxation during twitch contractions, which is consistent with a greater Ca2+-sensitivity of mature muscle. Because it becomes Key words: insect, muscle, ontogeny, dragonfly, Libellula pulchella, Odonata, phenotypic plasticity, troponin T, Ca2+-sensitivity, twitch, activated more quickly and relaxes more slowly, mature contraction kinetics. flight muscle is able to generate, with each twitch, more
Introduction Significant behavioral and thermoregulatory changes occur during adult maturation of Libellula pulchella dragonflies. Newly emerged adults (tenerals) are relatively sedentary, spending less than 2 % of their time in flight, whereas these dragonflies are much more active at maturity, spending an average of 30 % of certain periods of the day in flight, much of which is strenuous (Marden et al. 1996). Thoracic temperature (Tth) during flight also varies with age, as tenerals have Tth values ranging from 29 to 40 °C, whereas matures have Tth values ranging from 38 to 45 °C (Marden et al. 1996). The thermal physiology of flight performance undergoes a parallel change, as matures have a mean optimal Tth (the temperature at which maximum lift is produced) of 43.6 °C compared with the teneral mean optimal Tth of 34.6 °C (Marden, 1995a). At high Tth, matures generate an average of 50 % more lift per kilogram of flight muscle and have greater wingbeat frequencies than tenerals. The thermal shift and the marked increase in flight performance evident during maturation of L. pulchella *e-mail:
[email protected].
dragonflies could have a wide variety of mechanistic bases. We hypothesized that the mechanism might involve muscle contraction, and we therefore set out to examine two fundamentally different aspects of muscle contractile physiology: (1) crossbridge kinetics, as shown by shortening velocity and tetanic tension of intact muscle, and (2) contractile regulation by thin filament proteins, as shown by the Ca2+sensitivity of skinned muscle fibers. The results presented here suggest that a change in isoform expression of the Ca2+ regulatory protein troponin T causes a substantial increase in Ca2+-sensitivity of muscle activation which, in turn, affects the timing and force generation of twitch contractions, which ultimately determine whole-organism performance. Materials and methods Dragonflies and their ages Libellula pulchella Drury dragonflies (Odonata, Libellulidae) were collected at field sites in Centre County, Pennsylvania,
1474 G. H. FITZHUGH AND J. H. MARDEN USA. Relative age, broadly classified as teneral (newly emerged), adolescent (approximately midway between teneral and mature in body mass) or mature, was determined according to objective criteria described previously (Marden, 1995a). Note that flight muscle mass approximately doubles between emergence and maturity (Marden, 1995a) as a result of hypertrophy of muscle cells (Marden, 1989). Whole-muscle contractile performance Shortening velocity, tetanic tension and twitch contraction kinetics were measured from in situ preparations of individual mechanically isolated L. pulchella flight muscles. The head, wings and legs were removed (the abdomen was left in place to provide ventilation via its continued rhythmic contractions), after which the ventral thorax was mounted with epoxy resin to the bottom of a temperature-controlled chamber in such a way that the dorso-ventral flight muscles were oriented vertically. The first dorso-ventral muscle of the mesothorax, which drives the downstroke of the forewing leading edge, was isolated through a small incision in the dorsal thoracic cuticle, and its apodeme was attached with cyanoacrylate glue to an insect pin suspended from the lever arm of a Cambridge Technology 300B lever system. This muscle was held at a length approximately 2 % longer than its resting length, as determined by comparison with the undisturbed contralateral muscle. A 0.25 ms square-wave stimulus (a single stimulus for twitches; a 200 Hz train for tetani) from a Grass S4B stimulator was applied with fine-gauge electrodes inserted in the lateral thorax; the intensity of the stimulus was set at 125 % of supramaximal voltage. Muscle temperature was monitored with a fine-gauge copper–constantan thermocouple inserted into the thorax and connected to a Physitemp Bat-12 thermocouple thermometer. Water was pumped from a temperature-controlled bath through the water jacket of the test chamber in order to maintain muscle temperature at desired levels. A MacLab/8 (AD Instruments) was used to convert force and position signals from the lever system into 12-bit digital data and send them to a Macintosh Quadra 700, where they were monitored in real time with software emulating an oscilloscope (Scope; AD Instruments) at a sampling frequency of 4 kHz. Shortening velocity and tetanic tension were measured for one muscle preparation from each of 10 tenerals and 12 matures. Muscles were tested at 1–6 temperatures (alternating between either an ascending or a descending order of temperatures at 3–5 °C intervals from a starting point at room temperature). Experiments were terminated whenever tetanic tension degraded by more than 25 % from levels measured at the start of the experiment. This standard of preparation integrity is more lenient than that typically used for studies of insect muscle (Stevenson and Josephson, 1990; Marden, 1995b) because our preparations showed a poor capacity to maintain their original tension production. However, shortening velocity was not affected by tetanic tension independently of temperature (P=0.27). We therefore assumed that force degradation was a result of decreasing neural or
synaptic function, which resulted in submaximal recruitment of motor units, whereas crossbridge cycling of the active motor units was apparently not affected adversely. At the conclusion of experiments, cuticle and tissue surrounding the isolated muscle were dissected away, and the resting length of the muscle was measured to the nearest 0.1 mm using digital calipers. Muscle mass was measured to the nearest 0.1 mg using a Mettler balance. Cross-sectional area was estimated as the ratio of muscle mass to length. At each experimental temperature, shortening velocity was measured from the first 1 ms of length change following the establishment of a stable tension during isotonic contraction (see Marden, 1995b, for details). Maximum shortening velocity (Vmax) was determined from a curve fitted to velocity as a function of relative tension using a hyperbolic–linear equation (Marsh and Bennett, 1986; performed iteratively using an Igor software routine supplied by R. L. Marsh). Estimates of shortening velocity (m s−1) and tension (N) at the tension yielding maximum power output (as determined by the hyperbolic–linear equation) were multiplied together and divided by fresh muscle mass (kg) in order to derive maximum instantaneous power output (W kg−1). Measurements of twitch contraction kinetics were performed using the same arrangement as described above with a completely separate set of preparations wherein muscles were held at constant length and excited with single rather than multiple stimuli. These muscles were cooled to a temperature of approximately 16 °C, and the temperature was then raised slowly (approximately 0.5 °C min−1) until there was no longer a response to stimulation. Twitches were stimulated at least every 30 s and recorded at approximately 0.5 °C intervals. Twitch data were collected from five tenerals, three adolescents and seven matures. For each twitch, the time to peak tension, the time to halfrelaxation and the maximal force were determined. Temperatures were truncated to whole degrees, and data were pooled by age group within each temperature. Skinned fiber experiments Flight muscles from L. pulchella dragonflies were dissected and demembranated on ice for 1 h in an isotonic relaxing solution (20 mmol l−1 imidazole, 3 mmol l−1 MgATP, 5 mmol l−1 creatine phosphate and 5 mmol l−1 EGTA at pH 7, pCa 8 and 180 mmol l−1 total ionic strength; ionic strength adjusted using potassium methane sulfonate; Andrews et al. 1991) containing 0.5 % Triton X-100. The skinned fibers were used immediately or stored at −20 °C in 1:1 glycerol:relaxing solution for up to 3 weeks. Solutions of similar ionic composition (pH 7 at 25 °C) without detergent were prepared at pCa 8, 7, 6.5, 6, 5.75, 5.5, 5.25, 5, 4.5 and 4, using the computer program BES_BATH (Godt and Lindley, 1982). As temperature was raised or lowered from 25 °C during experiments, the pH of these solutions varied according to the pKa of the imidazole buffer, thereby closely approximating natural temperature-induced changes in pH of the intracellular milieu (Somero, 1986). The
Dragonfly muscle 1475 pCa of these solutions also varied as temperature was raised or lowered from 25 °C, but this effect was small, changing only by a few hundredths of a unit at the most extreme temperatures. A bundle of approximately 4–7 fibers was held at each end with a clamp made from a 23 gauge syringe needle and a 0.29 mm insect pin. The syringe needle was blunt-cut to a length of 1.5 cm, and the barrel was ground half-way through along 5 mm of its length, leaving a semi-circular trough at one end of the needle. One end of the muscle was laid in the trough and clamped by a length of pin which slid into the syringe needle beyond the trough and was glued in place with cyanoacrylate glue. This clamp is a modification of one described by Moss (1979). One clamp was mounted to a fixed point within a temperature-controlled chamber filled with relaxing solution. The other clamp, also within the chamber, was attached to the lever arm of a Cambridge Technology 300B lever system (which simultaneously measures both length and tension). The MacLab/8 converted force and position signals from the lever system into 12-bit digital form and sent them to a Macintosh Quadra 700, where they were monitored in real time with software emulating a chart recorder (Chart; AD Instruments) at a sampling frequency of 20 Hz. A length–tension curve was generated for the fiber bundle in activating solution (pCa 5.5; this Ca2+ concentration was chosen because the muscle did not recover well from prolonged immersion in solutions of higher Ca2+ concentrations), and the muscle was fixed at a length just shorter than the length at which the tension started to decrease from its maximum. The length–tension curve for an individual fiber bundle was highly reproducible, provided the bundle was not stretched excessively beyond its point of maximum tension. This method fixes sarcomere length at maximum crossbridge overlap (Moss, 1979). Samples of teneral and mature fibers were fixed with glutaraldehyde while mounted at the point of maximal tension; when sectioned longitudinally and examined with a transmission electron microscope, they showed nearly uniform sarcomere lengths (2.2–2.4 µm). As the length of the fiber bundle was held constant, its isometric tension was measured at various Ca2+ concentrations and temperatures. Data were first collected at 20 °C, and the fiber bundle was then subjected to temperatures of 15, 25, 30, 35 and 40 °C. Some fibers did not survive the entire range of temperatures (they no longer generated measurable tension changes between relaxation and full activation, or they tore). For some dragonflies, we tested more than one fiber in order to cover nearly all of the experimental temperatures. An average of 5.5 pCa50 estimates (described below) per individual dragonfly were obtained (S.D.=1.3; N=6 tenerals and N=7 matures). All activating solutions were used at each temperature; they were added in order of increasing Ca2+ concentration (a slightly different data set was generated if the solutions were added in reverse order). The chamber and fiber were rinsed three times with each new activating solution prior to its final addition. Tension was allowed to equilibrate at each temperature and Ca2+ concentration prior to data collection.
For each skinned fiber preparation, relative tension at each point on a pCa series (the full complement of activating solutions run at a given temperature) was determined as the ratio of tension to the range of tension over that same pCa series. The pCa50 (the Ca2+ concentration at which the fiber bundle was half-maximally activated) was determined using an Igor software routine that performed an iterative fit of the Hill equation (Hill, 1910; Godt and Lindley, 1982; Andrews et al. 1991) to measurements of percentage maximal tension versus Ca2+ concentration. SDS–PAGE gels and western blots Flight muscle was dissected from the thorax of freshly killed L. pulchella dragonflies and demembranated on ice for 1 h in the same detergent solution as described above for preparing skinned fibers. This treatment removed most of the watersoluble and membrane-bound proteins, leaving a fraction enriched in contractile proteins. Approximately 5 mg of washed flight muscle was placed in 200 µl of SDS reducing buffer (62.5 mmol l−1 Tris, pH 6.8, 10 % glycerol, 2 % SDS, 0.5 % 2-β-mercaptoethanol and 0.0025 % Bromophenol Blue) and frozen until used. Polyacrylamide gels (16 cm×16 cm; 0.75 mm thick) were cast and run using BioRad PROTEAN II xi electrophoresis equipment. The running gel contained 12 % polyacrylamide, 0.1 % SDS and 375 mmol l−1 Tris, pH 8.8, and was polymerized by the addition of 0.05 % ammonium persulfate and 0.03 % TEMED (N,N,N′,N′Tetramethylethylenediamine) (Laemmli, 1970). The stacking gel contained 4 % polyacrylamide and 125 mmol l−1 Tris, pH 6.8. Frozen samples in reducing buffer were heated to 95 °C for 5 min and centrifuged at 14 000 revs min−1 for 10 min before use. A buffer volume containing soluble protein from 0.25 mg of flight muscle (approximately 10 µl) was loaded onto the gel. (Gels run with ground portions of the muscle in reducing buffer contained the same types and proportions of proteins but produced greater background staining.) The molecular mass standards used were Perfect Protein markers from Novagen, recombinant proteins of constant amino acid composition at specified molecular masses. Gels were run at 17 °C in a buffer of 0.1 % SDS, 0.025 mol l−1 Tris and 0.2 mol l−1 glycine. A constant (40 mA) current was applied using a BioRad model 200/2.0 power supply. The protocol and reagents supplied with the BioRad silver stain plus kit were used to silver stain the gels. Proteins from gels were transferred to nitrocellulose using standard blotting methods (Towbin et al. 1979) with the BioRad trans-blot cell and the model 200/2.0 power supply. The blotting buffer contained 10 mmol l−1 Tris, pH 8.0, 150 mmol l−1 NaCl and 0.05 % Tween 20. Monoclonal antibodies to troponin T (MAC 145; Bullard et al. 1988), troponin C (MAC 352), troponin H and troponin I (MAC 143, which binds to both H and I; all antibodies provided by B. Bullard) were applied to the nitrocellulose after nonspecific blocking of the membrane using 3 % bovine serum albumin (BSA) and conjugated with a goat anti-rat alkaline phosphatase
1476 G. H. FITZHUGH AND J. H. MARDEN secondary antibody (Sigma). The nitrocellulose was then stained using the BioRad immuno-blot alkaline phosphatase assay kit. Duplicate sets of lanes were run on each half of a single gel and blotted onto nitrocellulose. The nitrocellulose was then cut and one set of lanes was developed for the antibody, while the other set of lanes was stained with the BioRad colloidal gold total protein stain kit. This procedure provided confirmation of the precise location of the troponin bands on the gel.
50 mN
Length 10 % rest length
Tetanic tension (N cm−2)
(musclelengths lengths s−1) VVmax max (muscle
20 ms
(W kg −1) Power output (W
Results Whole-muscle experiments The contractile characteristics of L. pulchella whole-muscle preparations were similar to those observed in previous studies of synchronous insect flight muscle. Tetanic tension averaged 10–12 N cm−2, which is very similar to the range of values reported for katydid metathoracic flight muscles (Josephson, 1984) and flight muscles of geometrid moths (Marden, 1995b). If an adjustment is made for the 46 % of cross-sectional area in dragonfly muscle that is occupied by mitochondria (Marden, 1989), these tetanic tensions compare favorably with values obtained from vertebrate striated muscle (15–30 N cm−2; Close, 1972). Maximal shortening velocities of 8–10 muscle lengths s−1 (L s−1) are consistent with previous warm-temperature data from locusts and katydids (5–16 L s−1; Josephson, 1984) and moths (8–10 L s−1; Marden, 1995b). Thus, our dragonfly whole-muscle preparations appeared to be functionally competent, despite their somewhat accelerated rates of performance decay. Maximum shortening velocity, tetanic tension and instantaneous power output during isotonic contraction did not vary as a function of age or age×temperature interaction (Fig. 1; Table 1). In contrast, twitch contraction kinetics varied significantly with age. Fig. 2 shows plots of time to peak tension (TTP; the interval between stimulus onset and peak tension) and time to half-relaxation (THR; the interval between peak tension and half-maximal tension during the relaxation phase) versus temperature for teneral and mature muscle. Mature muscle showed lower TTP and higher THR values at all but the very highest experimental temperatures, where teneral muscle was rapidly degenerating. A linear transformation of these data into logTTP and logTHR versus log(temperature) was made prior to statistical analysis. The linear range of the transformed data extended from 18 to 38 °C; data outside that range were excluded. A split-plot analysis of variance (ANOVA), controlling for differences among individuals that were tested repeatedly over variable temperatures, was performed to determine whether logTTP is affected by age and temperature. A similar split-plot ANOVA (Table 2) was performed with the response variable logTHR. All of the effects tested were highly significant (P
