Acidic and basic troponin T isoforms in mature fast-twitch skeletal muscle and effect on contractility

Ozgur Ogut1, Henk Granzier2, and Jian-Ping Jin1

1 Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4970; and 2 Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology, Washington State University, Pullman, Washington 99164-6520


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Developmentally regulated alternative RNA splicing generates distinct classes of acidic and basic troponin T (TnT) isoforms. In fast-twitch skeletal muscles, an acidic-to-basic TnT isoform switch ensures basic isoform expression in the adult. As an exception, an acidic segment in the NH2-terminal variable region of adult chicken breast muscle TnT isoforms is responsible for the unique exclusive expression of acidic TnTs in this muscle (O. Ogut and J.-P. Jin. J. Biol. Chem. 273: 27858-27866, 1998). To understand the relationship between acidic vs. basic TnT isoform expression and muscle contraction, the contractile properties of fibers from adult chicken breast muscle were compared with those of the levator coccygeus muscle, which expresses solely basic TnT isoforms. With use of Triton X-100-skinned muscle fibers, the force and stiffness responses to Ca2+ were measured. Relative to the levator coccygeus muscle, the breast muscle fibers showed significantly increased sensitivity to Ca2+ of force and stiffness with a shift of ~0.15 in the pCa at which force or stiffness was 50% of maximal. The expression of tropomyosin, troponin I, and troponin C isoforms was also determined to delineate their contribution to thin-filament regulation. The data indicate that TnT isoforms differing in their NH2-terminal charge are able to alter the sensitivity of the myofibrillar contractile apparatus to Ca2+. These results provide evidence linking the regulated expression of distinct acidic and basic TnT isoform classes to the contractility of striated muscle.

alternative ribonucleic acid splicing; developmental regulation; calcium; activation of force and stiffness; tropomyosin


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

TROPONIN T (TnT) is the tropomyosin (Tm)-binding subunit of the troponin complex and a central element in the thin-filament-linked Ca2+ regulatory system of vertebrate striated muscle (20). A large diversity of TnT isoforms is expressed in striated muscles as a result of alternative RNA splicing (for recent review see Ref. 31). The 5'-variable region of the TnT transcript is responsible for multiple isoforms that differ in their NH2-terminal primary structure (3, 7, 14, 19, 35, 36, 40). In addition, alternative splicing of the mutually exclusive exons 16 and 17 generates additional isoforms from the fast-twitch skeletal muscle TnT gene (3, 36, 40, 43). Although the alternatively spliced NH2-terminal variable region (13, 29) is quantitatively acidic in all TnTs, the expression of alternatively spliced exons results in TnT isoforms with a wide range of overall NH2-terminal charge. A common theme in the pattern of TnT isoform expression during cardiac and skeletal muscle development is the regulated high-to-low molecular weight (Mr) and acidic-to-basic isoform switch (16, 40). The functional significance of the switch between TnT isoform classes of distinct physical properties remains largely unknown because of the isoform diversity, which complicates the characterization of individual TnTs. Nonetheless, differences in the Ca2+ sensitivity of the actomyosin-ATPase were demonstrated in reconstituted systems containing two bovine cardiac TnT isoforms with differences in NH2-terminal size and charge (39). Studies have correlated TnT isoform expression with muscle contractility in normal and pathological states (1, 2, 34), although these investigations have not delineated the physical properties of the TnT isoforms that change in expression level. While the NH2-terminal variable region appears to be nonessential for TnT's core function in actomyosin activation (28), we have demonstrated that the NH2-terminal structure is able to modulate TnT's conformation and interaction with other thin-filament proteins (25, 41). Furthermore, acidic and basic fast-twitch skeletal muscle TnT isoforms have differences in their ability to bind Tm and troponin I (TnI) in response to decreased pH, indicating that the NH2 terminus of TnT may contribute to the tolerance of muscle to acidosis (26).

The physiological significance of acidic and basic TnT isoforms needs to be further characterized in an integrated muscle system. In the present study we have investigated the relationship between the expression of acidic and basic fast TnT isoforms and the contractility of muscle. Using skinned fibers from adult chicken breast muscle (exclusive acidic TnT expression) and levator coccygeus (exclusive basic TnT expression), we show that acidic TnT expression contributed to the function of the contractile apparatus by sensitizing force and stiffness responses to Ca2+. Therefore, the increased expression of basic TnT isoforms in adult muscle may contribute to the lower sensitivity to Ca2+ than in neonatal muscle (37), implying an important modulatory role for TnT isoforms in the fine tuning of muscle contraction.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation of muscle homogenates. Adult White Leghorn chicken (Gallus domesticus) muscles were identified according to Nickel et al. (24) and excised. A sample (~100 mg) of the fresh muscle was immediately homogenized in 1 ml of SDS-PAGE sample buffer containing 1% SDS and heated to 80°C for 5 min. The total protein extracts were clarified by centrifugation at 14,000 g for 5 min in a microcentrifuge before SDS-PAGE.

Western blot analysis of TnT, Tm, and TnI isoform expression. The 1% SDS extracts of muscles were resolved by two SDS-PAGE systems to maximize the resolution of protein isoforms: 1) 12% Laemmli SDS-PAGE with an acrylamide-to-bisacrylamide ratio of 29:1 or 2) 14% Laemmli SDS-PAGE with an acrylamide-to-bisacrylamide ratio of 180:1. In 29:1 12% SDS-PAGE, low-Mr TnT isoforms are well separated, but high-Mr TnT isoforms comigrate with actin, causing a broadening of these TnT bands in Western blots. In 180:1 14% SDS-PAGE, high-Mr TnT isoforms are well resolved and separated from the actin band, but stacking of low-Mr TnT isoforms may occur. Resolved proteins were transferred to 0.45-µm nitrocellulose membranes, as previously described (25). Replica nitrocellulose membranes were incubated at 4°C overnight with rabbit polyclonal antisera raised against chicken breast muscle TnT (RATnT) (41) or a chicken fast TnT NH2-terminal peptide (Tx) conjugated to keyhole limpet hemocyanin (RATx) (18), an anti-Tx monoclonal antibody (MAb) 6B8 (41), a chicken fast-twitch skeletal muscle TnT-specific MAb 3E4 (41), a cardiac muscle TnT-specific MAb CT3 (17), an alpha - and beta -isoform-specific anti-Tm MAb CH1 (22) (provided by Dr. J. J.-C. Lin, University of Iowa), or a TnI-specific MAb TnI-1 (J.-P. Jin and F. Yang, unpublished results). The subsequent washing, incubation with alkaline phosphatase-labeled anti-mouse or rabbit IgG secondary antibody (Sigma Chemical), and 5-bromo-4-chloro-3-indolylphosphate-nitro blue tetrazolium color development were performed as previously described (25).

Purification and identification of troponin C. To determine troponin C (TnC) isoform expression, a recombinant prokaryotic expression plasmid encoding chicken fast-twitch skeletal muscle TnC (kindly provided by Dr. L. B. Smillie, University of Alberta) was used to express TnC in Escherichia coli BL21(DE3)pLysS. The culture medium, growth, and induction conditions have been previously described (25). The culture was induced by 0.2 mM isopropyl-1-thio-beta -D-galactopyranoside at an optical density at 600 nm of 0.9 and grown for another 3 h. After the induced bacterial culture was harvested, the cells were resuspended in 6 M urea, 30 mM Tris · HCl, pH 8.0, and 2 mM MgCl2 and lysed by three passes through a French press at 500-700 psi. The clarified lysate was loaded onto a DE52 ion-exchange column equilibrated in the same buffer and eluted by a 0-300 mM KCl gradient. The fractions containing the TnC peak were identified by SDS-PAGE, collected, and dialyzed for two changes against 4 liters of double-distilled water. After lyophilization the protein powder was resuspended in a minimal volume of 0.5 M KCl, 20 mM Tris · HCl, pH 8.0, and 2 mM MgCl2 and resolved by a gel filtration column (Sephadex G75, Pharmacia-Amersham). The TnC peak from the gel filtration column was identified and dialyzed as described above before lyophilization for long-term storage at -20°C. With the purified chicken fast-twitch skeletal muscle TnC as an immunogen, a mouse antiserum (MATnC) was raised and used to identify TnC isoforms in Western blots of fiber homogenates.

To provide a native fast-twitch skeletal muscle TnC control, TnC was also purified from chicken breast muscle, as described by Potter (32). To monitor the difference in migration between the skeletal and cardiac TnC isoforms, a prokaryotically expressed mouse cardiac TnC protein was used. E. coli BL21(DE3)pLysS was transformed with a pET3d (Novagen) expression plasmid encoding mouse cardiac TnC (A. Chen and J.-P. Jin, unpublished results), cultured, and induced with isopropyl-1-thio-beta -D-galactopyranoside, as described above, to express cardiac TnC. Total protein extracts from the bacterial cultures were prepared for SDS-PAGE, as described previously (25). To maximize the separation of TnC isoforms by SDS-PAGE, 15% Laemmli gel with an acrylamide-to-bisacrylamide ratio of 29:1 was used.

Preparation of skinned chicken muscle fibers. Fiber bundles were dissected from the pectoralis major and levator coccygeus muscles of adult White Leghorn chickens after their euthanization by CO2 inhalation. The bundles were skinned for 60 min at room temperature in relaxing solution [40 mM imidazole, 10 mM bis-(aminoethyl)glycolether-N,N,N',N'-tetraacetic acid, 6.4 mM magnesium acetate, 5.9 mM ATP sodium salt, 5 mM NaN3, 80 mM potassium proprionate, 10 mM creatine phosphate, 1 mM dithiothreitol, 0.04 mM leupeptin, 0.5 mM phenylmethylsulfonate, pH 7.0] that contained 1% (wt/vol) Triton X-100 (catalog no. 28314, Pierce Chemical). The bundles were then washed twice with relaxing solution, twice with relaxing solution in 50% glycerol and then stored at -20°C in relaxing solution in 50% glycerol. The bundles were used within 2 wk.

Mechanical measurements. The computer-controlled mechanics workstation used in this study has been described in detail by Granzier and Irving (11). Briefly, a small 100-µl chamber was mounted on an x-y stage of an inverted microscope that also contained a servomotor (model 6800, Cambridge Technology; step response ~0.3 ms, root mean square position noise ~0.5 µm) and a force transducer (model AME 801 E, Horton) with a strain gauge-conditioning amplifier (model 2310, Measurement Group, Raleigh, NC) that had a bandwidth of direct current of 10 kHz and a force resolution of ~100 µg. In addition to force, high-frequency stiffness was measured using 0.1% amplitude sinusoidal oscillations at 2.2 kHz. Force and stiffness results were expressed per unit cross-sectional area. The cross-sectional areas of the fibers were calculated from their measured maximal and minimal diameters, with the assumption of an elliptical cross section (11).

Single fibers were dissected from fiber bundles in relaxing solution-50% glycerol. The fibers were then transferred to the microscope, and the ends were wrapped around fine pins (100 µm diameter) that had been glued to the motor and the force transducer. To limit stretching of the wrapped portion of the fibers during a contraction, a small volume (<1 µl) of 2% glutaraldehyde was added to the wrapped portion of the fiber at the back side of the pin (5). The fiber was quickly immersed into the chamber that was being rapidly flushed with relaxing solution. The chamber was connected to a system allowing continual perfusion of the muscle fibers with relaxing solution or activating solution (pH of solutions = 7.0). The pCa of the perfusing solution was varied by mixing relaxing solution with various amounts of activating solution that contained the same components as the relaxing solution, with the addition of Ca2+ to 10 mM (8). In these experiments the filament lattice spacing was not controlled. However, no changes in fiber diameter were noticed as the pCa of the perfusing solution was varied. The chamber contained a small J-type thermocouple and was temperature controlled to 22°C in all experiments.

Sarcomere length measurement. Sarcomere length was measured with laser diffraction with use of an He-Ne laser beam focused to a diameter of ~250 µm. The diffraction pattern was collected with a bright-field objective (ELWD plan 40/0.55, Nikon); a telescope lens was focused on the back focal plane of the objective, and the diffraction was projected, after compression with a cylindrical lens, onto a photodiode array (model RL 256 C/17, Reticon). The first-order diffraction peak position was obtained (12) using a digital spot-position detector board (Dept. of Bioengineering, University of Washington, Seattle, WA) installed in an IBM AT computer. This signal was converted to sarcomere length by using a calibration curve that was established with the diffraction peaks of a 25-µm grating present in the chamber. Sarcomere length noise (peak to peak) was ~10 nm. Sarcomere length was measured in the central region of the muscle fibers and was 2.23 ± 0.10 and 2.28 ± 0.06 (SD) µm during the steady force plateau of contractions of the pectoralis major (n = 26) and levator coccygeus (n = 40) fibers, respectively.

After the mechanical measurements, cross sections from the fiber bundles were homogenized in SDS-PAGE sample buffer, resolved by SDS-PAGE, and immunoblotted using RATnT, 6B8, TnI-1, MATnC, and CH1 antibodies to verify the thin-filament protein isoforms in the fibers of chicken pectoralis and levator coccygeus muscles.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Identification of thin-filament protein isoforms in adult chicken muscles. Using a panel of specific polyclonal and monoclonal antibodies, we identified the TnT, TnI, TnC, and Tm isoforms expressed in representative adult chicken striated muscles. The expression patterns of fast-twitch skeletal muscle-specific (breast and gastrocnemius), slow-twitch skeletal muscle-specific (trapezius), and cardiac muscle-specific (left ventricle) thin-filament regulatory proteins were determined (Fig. 1). In fast-twitch skeletal muscle, immunoblots with RATnT show that a heterogeneity of fast-twitch skeletal muscle TnT isoforms is expressed, although these are present as two groups that differ by Mr. MAb 6B8 staining for the acidic NH2-terminal Tx segment indicates that the high-Mr isoform had an acidic NH2 terminus compared with the low-Mr counterparts, which are the normal basic adult isoforms (26, 40). In contrast, a single TnT isoform is identified by RATnT in the slow-twitch trapezius muscle. Western blotting with the anti-cardiac TnT MAb CT3, which cross-reacts with slow- but not fast-twitch skeletal muscle TnT (14), indicates that the only TnT isoform expressed in trapezius muscle is slow-twitch skeletal muscle TnT (44). Interestingly, the RATnT antiserum generated against chicken breast muscle TnT cross-reacts less with slow-twitch skeletal muscle TnT than with cardiac muscle TnT (Fig. 1), given that comparable amounts of muscle protein extracts were loaded as normalized by the actin bands. This indicates the presence of unique epitopes and the structural divergence of slow-twitch skeletal muscle TnT vs. its fast-twitch skeletal and cardiac muscle counterparts. Fast-twitch (lower Mr) and slow-twitch (higher Mr) skeletal muscle TnI isoforms were identified in the gastrocnemius and trapezius, respectively. A single cardiac muscle TnI isoform was expressed in the adult heart, and its migration was similar to slow-twitch skeletal muscle TnI in the 180:1 14% gel. In contrast, SDS-PAGE of mammalian cardiac and slow-twitch skeletal muscle TnI isoforms show significant differences in their apparent Mr (42). Tm isoforms were mixed alpha - and beta -Tm in the gastrocnemius and trapezius muscles but exclusively alpha -Tm in the heart and the breast muscles.


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Fig. 1.   Western blot identification of troponin T (TnT), tropomyosin (Tm), and troponin I (TnI) isoforms. Specific antibodies were used to determine TnT, Tm, and TnI isoforms expressed in adult chicken striated muscles. Shown together with SDS-PAGE of comparatively loaded samples, a variety of TnT isoforms are detected by a rabbit polyclonal antiserum raised against chicken breast muscle TnT (RATnT). CT3 monoclonal antibody (MAb) staining shows that, in trapezius and heart, only slow-twitch skeletal and cardiac muscle TnT isoforms are expressed, respectively. Multiple fast-twitch skeletal muscle TnT (fTnT) isoforms are present in fast-twitch skeletal muscles, as detected by MAb 3E4 in breast and gastrocnemius muscles. Anti-Tx MAb 6B8 was used to specifically detect expression of an NH2-terminal acidic stretch in breast muscle TnT. The alpha - and beta -isoforms of Tm were found, with breast muscle and heart expressing exclusively alpha -Tm. The fast-twitch skeletal muscle isoform of TnI (fTnI) was found in breast and gastrocnemius muscles, whereas slow-twitch skeletal muscle isoform TnI (sTnI) was expressed in trapezius and cardiac isoform TnI (cTnI) was expressed in heart. Gel concentration and acrylamide-to-bisacrylamide ratio used for SDS-PAGE are indicated. Mr, molecular weight (relative).

With use of the MATnC antiserum raised against chicken fast-twitch skeletal muscle TnC, TnC isoform expression was determined in cardiac and fast- and slow-twitch skeletal muscles (Fig. 2). The chicken breast and the levator coccygeus fibers expressed fast-twitch skeletal muscle TnC, indicated by comigration with fast-twitch skeletal muscle TnC expressed from the cloned cDNA or purified from chicken breast muscle. In contrast, the slow-twitch trapezius muscle and cardiac muscle expressed TnC isoforms of reduced mobility compared with fast-twitch skeletal muscle TnC. The chicken slow-twitch/cardiac muscle TnC migrates similarly to the mouse cardiac muscle TnC expressed from the cloned cDNA.


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Fig. 2.   Troponin C (TnC) isoform expression in striated muscles. A mouse antiserum raised against chicken breast TnC (MATnC) was used to determine TnC isoform expression in representative chicken striated muscles. With prokaryotically expressed or natively purified chicken breast muscle TnC used as controls, chicken breast and levator total homogenates are shown to express fast-twitch skeletal muscle TnC. Trapezius and cardiac muscles expressed higher-Mr TnCs, which migrated similarly to prokaryotically expressed mouse cardiac TnC.

The expression patterns of acidic and basic fast-twitch skeletal muscle TnT isoforms were determined by Western blotting with the RATnT and RATx antisera (Fig. 3). The inclusion of the Tx segment results in TnT isoforms with relatively acidic isoelectric points and higher Mr than their basic counterparts (Fig. 1) (26). The majority of the muscles in the pelvic limb of the chicken express basic TnT isoforms, consistent with fast TnT isoforms identified in other adult skeletal muscles (40). Therefore, the expression of acidic fast TnT isoforms in the adult chicken breast muscles provides a novel model to study the structure-function relationships of TnT.


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Fig. 3.   Distribution of acidic and basic fast TnT isoforms in adult chicken skeletal muscles. On total tissue homogenates from selected chicken skeletal muscles, patterns of total TnT isoform expression and acidic TnT isoform distribution were determined, respectively, by SDS-PAGE (12%, 29:1) and Western blotting with use of RATnT and RATx antisera. High-Mr, acidic TnTs (widened bands were due to overlap with actin band) were specifically expressed in pectoral limb muscles together with various amounts of basic fast-twitch skeletal muscle TnT isoforms. A notable exception was pectoralis superficialis (major), which expressed exclusively high-Mr, acidic fast-twitch skeletal muscle TnT isoforms. Conversely, the majority of muscles in the pelvic limb expressed low-Mr, basic TnT isoforms.

Developmental regulation of Tm expression. In contrast to other fast-twitch skeletal muscles, the adult chicken breast muscle expresses exclusively alpha -Tm. To determine whether the Tm expression pattern in the breast muscle is developmentally regulated, the Tm isoforms expressed in embryonic and adult breast major, gastrocnemius, and heart muscles were identified (Fig. 4). The gastrocnemius and heart show persistent mixed alpha - and beta -Tm and alpha -Tm expression, respectively, through development. Tropomyosin expression was mixed alpha  and beta  isoforms in the embryonic breast muscle, but unlike other fast-twitch skeletal muscles, the beta -Tm isoform was downregulated in favor of the exclusive expression of alpha -Tm in the adult. The developmental regulation of Tm isoforms in chicken breast muscle shows a correspondence to the increasing expression of acidic fast TnT isoforms in breast muscle during development (26).


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Fig. 4.   Developmental regulation of Tm isoforms in developing fast-twitch skeletal and cardiac muscles. Developmental expression of Tm was determined in breast major, gastrocnemius, and heart by Western blotting with use of 12% 29:1 SDS-PAGE on embryonic day 14 (ED14) and adult muscle protein extracts. Embryonic breast muscle expressed a mixture of alpha - and beta -Tm, similar to embryonic gastrocnemius, but beta -Tm expression was downregulated with development in favor of alpha -Tm. In contrast, there was no change in Tm expression pattern in gastrocnemius through development. Heart showed exclusive alpha -Tm expression from embryo to adult.

Ca2+ sensitivity of muscle fibers containing acidic or basic fast-twitch skeletal muscle TnT isoforms. To examine the relationship between TnT isoform expression and Ca2+ sensitivity, mechanical measurements were done with two representative, classical fast-twitch white skeletal muscles: the pectoralis major and the levator coccygeus. Both muscles produced high levels of isometric force in response to perfusion with activating solution (Fig. 5). The performance of the fibers was stable, and typically >10 contractions could be induced before a noticeable force decrease took place. To determine how much of the ends of the fibers had been fixed by glutaraldehyde, some of the fibers were activated at the end of the experiment and allowed to highly shorten by moving the motor closer to the force transducer. All sarcomeres were observed to shorten except those in a small region (~0.05-0.1 mm long) at each end of the fiber that had been fixed by glutaraldehyde. The fixed ends greatly limited end compliance. Because of end compliance, sarcomeres in the central region of fibers with unfixed ends may significantly shorten during activation, even though the length of the fiber is held constant; furthermore, the diffraction pattern of these sarcomeres often completely disappears. During our experiments, however, the diffraction pattern of fibers with ends that had been fixed remained very strong during activation and revealed that sarcomeres in the central region of the fiber shortened only a very short distance on activation: 43 ± 60 and 20 ± 43 (SD) nm for pectoralis major (n = 26) and levator coccygeus (n = 40), respectively.


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Fig. 5.   Contractile response of skinned pectoralis major and levator coccygeus (cocc) fibers. Representative tracings of response of sarcomere length, force, and stiffness of skinned pectoralis and levator fibers to Ca2+ at pCa 6.70 and 6.00 are shown. Higher response of pectoralis than of levator fibers at pCa 6.7 indicates higher Ca2+ sensitivity for force and stiffness.

The response of force and stiffness of the skinned pectoralis and levator muscle fibers to Ca2+ were measured. The Ca2+ sensitivities of force (P < 0.02) and stiffness (P < 0.01) were significantly higher in the pectoralis than in the levator fibers, as judged by the Ca2+ concentration required to achieve 50% of maximal response (Figs. 6 and 7, Table 1). There was no significant difference in the maximum force or stiffness per cross-sectional area generated by the pectoralis or levator coccygeus fibers, implying little difference in the maximum amount of cross bridges formed or the force per cross bridge, as estimated by dividing maximal force by maximal stiffness. Therefore, the Ca2+ sensitivity of force and stiffness in these muscles is reflective of regulation at the thin filament. Examination of the slopes of the Hill plots in Figs. 6 and 7 (Table 1) showed slightly lower cooperativity of Ca2+-activated force and stiffness development in the pectoralis fibers, although the differences were not statistically significant. It is interesting to note that stiffness exhibits greater Ca2+ sensitivity than force in pectoralis and levator fibers. A similar observation has been reported by Martyn and Gordon (23), who investigated force and stiffness in rabbit psoas fibers. Our results are consistent with their proposal that attached cross bridges exist in non-force-producing and force-producing states and that the relative population of these states is Ca2+ dependent. At low-to-intermediate Ca2+ levels the non-force-producing attached population is relatively large, explaining the high stiffness but low force levels.


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Fig. 6.   Force-pCa relationship of skinned pectoralis major and levator coccygeus muscle fibers. Pooled results and Hill fits of pooled data are shown. Insets: Hill coefficients and pCa at which force was 50% of maximal (pCa50); results were obtained from fitting each preparation separately and calculating mean ± SD of 4 pectoralis major and 5 levator coccygeus fibers. Although the lower Hill coefficient of pectoralis muscle was not statistically significant, pCa50 of force and stiffness are significantly higher for pectoralis than for levator coccygeus fibers. Maximal force at pCa 6.0 was 160 ± 30 kN/m2 for pectoralis major and 130 ± 14 kN/m2 for levator coccygeus fibers.



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Fig. 7.   Stiffness response of skinned pectoralis major and levator coccygeus muscle fibers. Pooled results and Hill fits from response of muscle stiffness to free Ca2+ for pectoralis major and levator coccygeus are shown. Pectoralis major fibers showed higher sensitivity of stiffness to free Ca2+, although maximal stiffness at pCa 6.0 of the 2 muscles was not different: 13.2 ± 7.1 mN/m2 for pectoralis major and 13.4 ± 2.6 mN/m2 for levator coccygeus fibers.


                              
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Table 1.   Contractility parameters of skinned pectoralis major and levator coccygeus muscles

The thin-filament protein isoforms in the muscle fibers prepared for contractility experiments were identified by immunoblotting with RATnT, 6B8, TnI-1, MATnC, and CH1 antibodies. The main difference between the two muscles was the exclusive expression of high-Mr acidic TnTs in the pectoralis fibers and the exclusive expression of low-Mr basic TnTs in the levator fibers (Fig. 8). Furthermore, the levator fibers showed heterogenous expression of alpha - (major) and beta  (minor)-Tm isoforms, whereas the pectoralis fibers showed predominantly alpha -Tm expression. TnI isoform expression was comparable, with a trace amount of slow-twitch skeletal muscle TnI expressed in the levator fibers. Both muscle fibers expressed fast-twitch skeletal muscle TnC.


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Fig. 8.   Thin-filament protein expression in skinned pectoralis and levator coccygeus fibers. Muscle samples used for skinned fiber experiments were homogenized in SDS-PAGE sample buffer and analyzed by Western blotting. To compare the 2 muscle samples, nitrocellulose filters were incubated with RATnT, 6B8, TnI-1, MATnC, or CH1 antibody to examine expression of TnT, TnI, TnC, and Tm isoforms. Levator coccygeus fibers showed expression of some beta -Tm and a trace amount of slow-twitch (S) skeletal muscle TnI in addition to alpha -Tm and fast-twitch (F) skeletal muscle TnI. TnC isoform expression was identical in both muscles. Cross-reactive bands, possibly myosin light chains, were also stained by polyclonal anti-TnC serum, likely because of structural homology to TnC (6). Pectoralis fibers expressed exclusively high-Mr, acidic fast-twitch skeletal muscle TnTs, whereas levator expressed low-Mr, basic fast-twitch skeletal muscle TnTs, accounting for the major difference between the 2 fibers.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Unique expression of acidic TnT isoforms in adult chicken fast-twitch skeletal muscle. The developmental acidic-to-basic TnT isoform expression switch is found in avian and mammalian cardiac and skeletal muscles. This observed shift is most dramatic in fast-twitch skeletal muscles, where the isoelectric points of the expressed TnT isoforms may increase from 5.04 in the neonate to 10.06 in the adult (Delta  = 5.02) (40). The importance of this large shift is not understood, since mouse cardiac muscle TnT isoform expression patterns show relatively modest shifts in their isoelectric point (Delta  = 0.27) (19). Our survey of adult chicken skeletal muscles demonstrated a heterogeneity of acidic and basic TnT expression. As shown previously, high-Mr fast-twitch skeletal muscle TnT isoforms in the mature chicken (pectoralis muscles) correspond to isoforms that are acidic compared with their low-Mr counterparts (26). The expression of acidic fast-twitch skeletal muscle TnTs in the adult chicken is unique, since all vertebrate animals examined express exclusively basic fast-twitch skeletal muscle TnT isoforms in adulthood (29, 40, 43). In chicken breast muscle the inclusion of an acidic NH2-terminal segment results in TnTs with significantly decreased isoelectric points (average 7.26) compared with those in the gastrocnemius muscle (average 8.47) (26). Although this segment is specific to the Galliformes pectoral muscles (18), its effect on TnT NH2-terminal size and charge is representative of the difference between TnT isoform classes during the acidic-to-basic switch in vertebrate skeletal muscle (26, 40). Therefore, chicken breast muscle may serve as a model system to determine the effects of acidic TnT isoform expression in the adult.

Effect of acidic fast-twitch skeletal muscle TnT isoforms on thin-filament Ca2+ sensitivity. The two fast-twitch skeletal muscles tested showed differences in the Ca2+ sensitivity of active force and stiffness. Among the thin-filament regulatory proteins, fast-twitch skeletal muscle TnI and TnC are expressed in both pectoralis and levator coccygeus fibers (Fig. 8). Apart from the acidic vs. basic TnT isoform expression, the two muscle types had differences in Tm isoform expression, with the pectoralis fibers expressing only alpha -Tm and the levator fibers expressing some beta -Tm in addition to alpha -Tm (Fig. 8). In previous studies, overexpression of beta -Tm relative to alpha -Tm in transgenic mouse hearts resulted in a slight sensitization of the force response to Ca2+ in skinned trabeculae (27). Because the levator coccygeus fibers show higher expression of beta -Tm but lower Ca2+ sensitivity for force and stiffness, Tm isoform expression cannot account for the difference in Ca2+ sensitivity. In agreement with this evidence, Reiser and co-workers (33) showed that although the majority of pectoralis single fibers expressed exclusively alpha -Tm, a minority of fibers expressed some beta -Tm in addition to alpha -Tm. Nonetheless, they found no differences in the Ca2+ sensitivity of force between these groups of fibers. Therefore, Tm isoform expression levels seem to play a minor role in the responses of the fibers in these mechanical measurements. We conclude that the differences in Ca2+ sensitivity between the levator and pectoralis fibers are likely due to TnT isoform expression, with higher Ca2+ sensitivity in muscles expressing acidic TnTs. This conclusion is consistent with observations that the Ca2+ response of force is more sensitive in neonatal than in adult striated muscles (10, 37), given that neonatal muscles express more acidic TnT isoforms. Therefore, the acidic-to-basic TnT isoform switch in developing avian and mammalian striated muscles (16, 19, 40) may contribute to a change in the Ca2+ sensitivity of thin-filament activation and the overall cooperativity of muscle contraction (Fig. 6).

It is known that alpha -Tm is usually the exclusive Tm isoform found in cardiac muscle (21), coexpressed with cardiac TnT, the most acidic of all TnT isoforms. It was shown that beta -Tm is downregulated during development of the breast muscle (Fig. 4), coincident with the upregulation of the expression of acidic fast-twitch skeletal muscle TnT isoforms (26). There is no change in Tm isoform expression during the development of the gastrocnemius, where only basic TnT isoforms are expressed. It is interesting to speculate that the programmed, exclusive expression of alpha -Tm in breast muscle may be a response to the acidic TnT isoform expression, possibly also contributing to the cooperativity of muscle activation seen in Fig. 6. Whether an evolutionary adaptation is responsible for the coexpression of specific TnT and Tm isoforms remains to be investigated.

Potential effect of the NH2-terminal charge of TnT isoforms on muscle contractility. Our data suggest that acidic and basic TnT isoforms may modulate the Ca2+ sensitivity of muscle contraction, implicating the NH2 terminus of TnT in this function. One possible mechanism for this change in sensitivity may be dictated by the NH2-terminal charge of TnT. By the Gibbs-Donnan equilibrium, acidic TnT isoforms with negative charges at the NH2 terminus may increase the local free Ca2+ concentration at the thin filament to a level higher than that in the bulk solution. In effect, this increased local Ca2+ concentration would be available to bind to TnC and trigger contraction. This would be relevant for both skinned and intact muscle fiber preparations. In this case, detailed experiments elucidating the molecular distance between the TnT NH2 terminus and the regulatory Ca2+-binding sites of TnC would determine the magnitude of this effect.

The effect of TnT isoforms on the actomyosin system may also be directly due to different interactions with the other components in the thin-filament regulatory assembly (39), such as Tm dimers, which, in a simple model, are believed to contribute to the steric block of F-actin-active sites and prevent myosin head attachment (for recent review see Ref. 38). The predominant structural difference among TnT isoforms lies in the NH2 terminus, a region that has been shown to interact with the head-to-tail overlap of Tm dimers (4). The NH2 and COOH termini of alpha - and beta -Tm show a conserved, high proportion of charged amino acids (Asp, Glu, His, Lys, Arg) (21), and the potential of ionic interactions between this region of Tm and the charged NH2 terminus of TnT is a potential mechanism through which TnT-Tm interactions may modulate the response of the thin filament to Ca2+ activation. Unique interactions between TnT and Tm isoforms have been shown in experiments by Pearlstone and Smillie (30), in which rabbit fast-twitch skeletal muscle TnT fragments showed different binding affinities to various tropomyosin isoforms. Furthermore, the variable NH2 terminus of TnT isoforms may dictate changes in the overall conformation and function of the protein (25, 41). More specifically, these differences in tertiary structure among TnT isoforms may affect their interaction with TnI, TnC, and Tm, providing another mechanism through which the regulated expression of TnT isoforms may affect thin-filament activation. Altogether, TnT may be a central molecule in modulating the function of the thin filament through expression of multiple classes of isoforms.


    ACKNOWLEDGEMENTS

We thank Jill Jin for the illustration of chicken muscle anatomy in Fig. 3, Dr. Larry Smillie for the TnC expression plasmid, and Dr. Jim Lin for the CH1 MAb.


    FOOTNOTES

This study was supported in part by grants from the Medical Research Council of Canada and the Heart and Stroke Foundation of Canada to J.-P. Jin and National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-42652 to H. Granzier.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: J.-P. Jin, Dept. of Physiology and Biophysics, Case Western Reserve University School of Medicine, 10900 Euclid Ave., Cleveland, OH 44106-4970 (E-mail: jxj12{at}po.cwru.edu).

Received 16 November 1998; accepted in final form 12 February 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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