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• W2139834186 abstract "In this study we investigated the physiological role of the cardiac troponin T (cTnT) isoforms in the presence of human slow skeletal troponin I (ssTnI). ssTnI is the main troponin I isoform in the fetal human heart. In reconstituted fibers containing the cTnT isoforms in the presence of ssTnI, cTnT1-containing fibers showed increased Ca2+ sensitivity of force development compared with cTnT3- and cTnT4-containing fibers. The maximal force in reconstituted skinned fibers was significantly greater for the cTnT1 (predominant fetal cTnT isoform) when compared with cTnT3 (adult TnT isoform) in the presence of ssTnI. Troponin (Tn) complexes containing ssTnI and reconstituted with cTnT isoforms all yielded different maximal actomyosin ATPase activities. Tn complexes containing cTnT1 and cTnT4 (both fetal isoforms) had a reduced ability to inhibit actomyosin ATPase activity when compared with cTnT3 (adult isoform) in the presence of ssTnI. The rate at which Ca2+ was released from site II of cTnC in the cTnI·cTnC complex (122/s) was 12.5-fold faster than for the ssTnI·cTnC complex (9.8/s). Addition of cTnT3 to the cTnI·cTnC complex resulted in a 3.6-fold decrease in the Ca2+ dissociation rate from site II of cTnC. Addition of cTnT3 to the ssTnI·cTnC complex resulted in a 1.9-fold increase in the Ca2+ dissociation rate from site II of cTnC. The rate at which Ca2+ dissociated from site II of cTnC in Tn complexes also depended on the cTnT isoform present. However, the TnI isoforms had greater effects on the Ca2+ dissociation rate of site II than the cTnT isoforms. These results suggest that the different N-terminal TnT isoforms would produce distinct functional properties in the presence of ssTnI when compared with cTnI and that each isoform would have a specific physiological role in cardiac muscle. In this study we investigated the physiological role of the cardiac troponin T (cTnT) isoforms in the presence of human slow skeletal troponin I (ssTnI). ssTnI is the main troponin I isoform in the fetal human heart. In reconstituted fibers containing the cTnT isoforms in the presence of ssTnI, cTnT1-containing fibers showed increased Ca2+ sensitivity of force development compared with cTnT3- and cTnT4-containing fibers. The maximal force in reconstituted skinned fibers was significantly greater for the cTnT1 (predominant fetal cTnT isoform) when compared with cTnT3 (adult TnT isoform) in the presence of ssTnI. Troponin (Tn) complexes containing ssTnI and reconstituted with cTnT isoforms all yielded different maximal actomyosin ATPase activities. Tn complexes containing cTnT1 and cTnT4 (both fetal isoforms) had a reduced ability to inhibit actomyosin ATPase activity when compared with cTnT3 (adult isoform) in the presence of ssTnI. The rate at which Ca2+ was released from site II of cTnC in the cTnI·cTnC complex (122/s) was 12.5-fold faster than for the ssTnI·cTnC complex (9.8/s). Addition of cTnT3 to the cTnI·cTnC complex resulted in a 3.6-fold decrease in the Ca2+ dissociation rate from site II of cTnC. Addition of cTnT3 to the ssTnI·cTnC complex resulted in a 1.9-fold increase in the Ca2+ dissociation rate from site II of cTnC. The rate at which Ca2+ dissociated from site II of cTnC in Tn complexes also depended on the cTnT isoform present. However, the TnI isoforms had greater effects on the Ca2+ dissociation rate of site II than the cTnT isoforms. These results suggest that the different N-terminal TnT isoforms would produce distinct functional properties in the presence of ssTnI when compared with cTnI and that each isoform would have a specific physiological role in cardiac muscle. During human heart development at least four isoforms of cardiac TnT (cTnT) 1The abbreviations used are: cTnT, cardiac troponin T; MOPS, 3-(N-morpholino)propanesulfonic acid; cTnC, cardiac troponin C; cTnI, cardiac troponin I; Tm, tropomyosin; Tn, troponin; ssTnI, slow skeletal troponin I; MHC, myosin heavy chain; DTT, dithiothreitol; fsTnT, fast skeletal TnT.1The abbreviations used are: cTnT, cardiac troponin T; MOPS, 3-(N-morpholino)propanesulfonic acid; cTnC, cardiac troponin C; cTnI, cardiac troponin I; Tm, tropomyosin; Tn, troponin; ssTnI, slow skeletal troponin I; MHC, myosin heavy chain; DTT, dithiothreitol; fsTnT, fast skeletal TnT. are expressed in a developmentally regulated manner (1Townsend P.J. Barton P.J. Yacoub M.H. Farza H. J. Mol. Cell. Cardiol. 1995; 27: 2223-2236Abstract Full Text PDF PubMed Scopus (84) Google Scholar, 2Anderson P.A. Malouf N.N. Oakeley A.E. Pagani E.D. Allen P.D. Circ. Res. 1991; 69: 1226-1233Crossref PubMed Scopus (307) Google Scholar). The expression level of the main fetal isoform, TnT isoform 1 (cTnT1), decreases and the expression of TnT isoform 3 (cTnT3) increases until cTnT3 becomes the only TnT isoform in the normal adult heart. Because TnT is one of the major regulatory proteins in the thin filament (3Zot A.S. Potter J.D. Annu. Rev. Biophys. Biophys. Chem. 1987; 16: 535-559Crossref PubMed Scopus (443) Google Scholar), isoform switching of TnT during heart development may be important in the Ca2+ regulation of myocardial contraction. Recently, we have shown that the human cTnT isoforms affect the Ca2+ sensitivity of force development and their ability to inhibit actomyosin ATPase activity in the presence of human cardiac troponin I (cTnI) (4Gomes A.V. Guzman G. Zhao J. Potter J.D. J. Biol. Chem. 2002; 277: 35341-35349Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Other groups have also shown that TnT isoforms affect the Ca2+ sensitivity of force development and ATPase activity (5Tobacman L.S. Lee R. J. Biol. Chem. 1987; 262: 4059-4064Abstract Full Text PDF PubMed Google Scholar, 6Tobacman L.S. J. Biol. Chem. 1988; 263: 2668-2672Abstract Full Text PDF PubMed Google Scholar, 7Reiser P.J. Greaser M.L. Moss R.L. J. Physiol. (Lond.). 1992; 449: 573-588Crossref Scopus (62) Google Scholar, 8Nassar R. Malouf N.N. Kelly M.B. Oakeley A.E. Anderson P.A. Circ. Res. 1991; 69: 1470-1475Crossref PubMed Scopus (97) Google Scholar, 9Ogut O. Granzier H. Jin J.P. Am. J. Physiol. 1999; 276: C1162-C1170Crossref PubMed Google Scholar). These results all suggest that the N terminus of TnT affects the physiological function of troponin (Tn). However, TnI isoforms also switch during the period of TnT isoform changes. Slow skeletal TnI (ssTnI) is the predominant TnI isoform throughout fetal life and gradually decreases during the first few months of postnatal development (10Sasse S. Brand N.J. Kyprianou P. Dhoot G.K. Wade R. Arai M. Periasamy M. Yacoub M.H. Barton P.J. Circ. Res. 1993; 72: 932-938Crossref PubMed Scopus (203) Google Scholar). Developmental down-regulation of ssTnI occurs even in the absence of cTnI. Mice lacking cTnI are born healthy, with normal heart and body weight, because ssTnI (the fetal troponin I isoform) compensates for the absence of cTnI until 15 days after birth when ssTnI expression declines steadily, resulting in TnI deficiency and death on day 18 (11Huang X. Pi Y. Lee K.J. Henkel A.S. Gregg R.G. Powers P.A. Walker J.W. Circ. Res. 1999; 84: 1-8Crossref PubMed Scopus (116) Google Scholar). The physiological relevance of cTnT isoforms in the human heart is still not well understood, and the physiological relevance of cTnT isoforms in fetal human cardiac Tn has not been investigated previously.Two recent reports (12Siedner S. Kruger M. Schroeter M. Metzler D. Roell W. Fleischmann B.K. Hescheler J. Pfitzer G. Stehle R. J. Physiol. (Lond.). 2003; 548: 493-505Crossref Scopus (133) Google Scholar, 13Metzger J.M. Michele D.E. Rust E.M. Borton A.R. Westfall M.V. J. Biol. Chem. 2003; 278: 13118-13123Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) suggest that the developmental changes in the Ca2+ sensitivity of force development originate primarily from the switch of ssTnI to cTnI. Incorporation of embryonic/fetal isoforms of Tm, TnT, and TnI into the adult sarcomere by using gene transfer suggested that TnI, but not Tm or TnT embryonic isoforms, influenced calcium regulation of contraction (13Metzger J.M. Michele D.E. Rust E.M. Borton A.R. Westfall M.V. J. Biol. Chem. 2003; 278: 13118-13123Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Another report (12Siedner S. Kruger M. Schroeter M. Metzler D. Roell W. Fleischmann B.K. Hescheler J. Pfitzer G. Stehle R. J. Physiol. (Lond.). 2003; 548: 493-505Crossref Scopus (133) Google Scholar) correlated the switching of TnT and TnI isoforms with the Ca2+ sensitivity of force generation in mice and, based on these results, suggested that the change in Ca2+ sensitivity is mainly due to TnI isoform switching. The main goal of the present study was to gain insight into the effect of the N-terminal hypervariable region in cTnT in the presence of ssTnI (fetal TnI) on cardiac muscle contraction to determine whether the cTnT isoforms would still have a functional role in the presence of ssTnI.Three cTnT isoforms (Fig. 1) were investigated using several in vitro functional assays to determine whether any differences exist between the adult isoform (cTnT3) and the other isoforms of cTnT. In reconstituted fibers containing the cTnT isoforms in the presence of ssTnI, cTnT1 containing fibers showed increased Ca2+ sensitivity of force development (ΔpCa50 = +0.17) and maximal force compared with cTnT4 containing fibers. These results suggest that cTnT isoforms are able to modulate Ca2+ sensitivity even in the presence of ssTnI. The ability of Tn complexes to inhibit ATPase activity was significantly lower for all cTnT isoforms in the presence of ssTnI than in the presence of cTnI. The rate at which Ca2+ is released from site II of cTnC in Tn complexes was affected by both the cTnT and TnI isoforms present. cTnT had a greater effect on the kinetics of Ca2+ release from site II of cTnC in the presence of cTnI than in the presence of ssTnI. The N-terminal alternatively spliced region of cTnT also affected the solubility of cTnT. Our results suggest that this alternatively spliced N-terminal region of cTnT is an important modulator of the Ca2+ sensitivity of force development in both fetal and adult heart muscle and also affects the maximal force and the maximal and the minimal actomyosin ATPase activity. The results also suggest that the isoform of TnI present is important for some of the functional differences between the cTnT isoforms.MATERIALS AND METHODSExpression and Purification of cTnT Isoforms—Human cTnT isoforms were made by using a sequential overlapping PCR method as described previously (14Ausubel F.M. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Janssen K. Current Protocols in Molecular Biology. John Wiley & Sons, New York1995: 8.0.1-8.5.9Google Scholar). Standard methods previously used in this laboratory were utilized for expression and purification of the different cTnT isoforms (4Gomes A.V. Guzman G. Zhao J. Potter J.D. J. Biol. Chem. 2002; 277: 35341-35349Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Briefly, bacterially expressed and extracted cTnTs were purified on a Fast Flow Q-Sepharose column. cTnT was eluted from this column with a NaCl gradient (0–0.5 m). The semi-purified cTnT was then further purified on a cTnC affinity column. cTnT was eluted from this column with a double gradient of urea and EDTA (0–6 m and 0–3 mm, respectively). All steps were performed at 4 °C unless otherwise indicated. The purity of the isolated cTnT isoforms (>95%) was determined by SDS-PAGE.Expression and Purification of ssTnI—ssTnI cDNA was cloned from a human library using standard techniques and verified by sequencing. Similar methods previously used in this laboratory for purification of cTnI were utilized for expression and purification of the ssTnI (4Gomes A.V. Guzman G. Zhao J. Potter J.D. J. Biol. Chem. 2002; 277: 35341-35349Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 15Szczesna D. Zhang R. Zhao J. Jones M. Guzman G. Potter J.D. J. Biol. Chem. 2000; 275: 624-630Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Briefly, bacterially expressed and extracted ssTnI was purified on a Fast Flow S-Sepharose column. ssTnI was eluted from this column with a NaCl gradient (0–0.5 m). The semi-purified ssTnI was then further purified on a cTnC affinity column. ssTnI was eluted from this column with a double gradient of urea and EDTA (0–6 m and 0–3 mm, respectively).Formation of the Troponin Complex—Formation of the human cardiac Tn complexes containing human recombinant cTnT, cTnC, and TnI was carried by a modified method of the one recently described by Szczesna et al. (15Szczesna D. Zhang R. Zhao J. Jones M. Guzman G. Potter J.D. J. Biol. Chem. 2000; 275: 624-630Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Proteins were first dialyzed against a solution containing 3 m urea and 1 m KCl and then in solution containing 0 m urea and 1 m KCl. Complexes were formed in the presence of 0 m urea and 1 m KCl and then dialyzed against successively lower concentrations of KCl as described previously (15Szczesna D. Zhang R. Zhao J. Jones M. Guzman G. Potter J.D. J. Biol. Chem. 2000; 275: 624-630Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Proper stoichiometry was verified by SDS-PAGE. Although not done routinely, gel filtration of these formed Tn complexes showed that this reconstitution method resulted in a single species.Actin-Tm-activated Myosin-ATPase Assay—Porcine cardiac myosin, rabbit skeletal F-actin, porcine cardiac Tm, and recombinant human cardiac TnC were prepared as described previously (11Huang X. Pi Y. Lee K.J. Henkel A.S. Gregg R.G. Powers P.A. Walker J.W. Circ. Res. 1999; 84: 1-8Crossref PubMed Scopus (116) Google Scholar, 12Siedner S. Kruger M. Schroeter M. Metzler D. Roell W. Fleischmann B.K. Hescheler J. Pfitzer G. Stehle R. J. Physiol. (Lond.). 2003; 548: 493-505Crossref Scopus (133) Google Scholar). The ATPase inhibitory assay was performed in a 1-ml reaction mixture of 100 mm KCl, 4 mm MgCl2, 1.0 mm EGTA, 2.5 mm ATP (Mg2+-ATP), 0.1 mm DTT, 10 mm MOPS, pH 7. The ATPase activation assay was carried out in the same 1-ml reaction mixture with 1 mm EGTA replaced with 0.5 mm CaCl2. F-actin (3.5 μm), myosin (0.6 μm), and Tm (1 μm) were homogenized and added to the reaction tube after the addition of buffer and Tn to the assay tube. Different Tn complexes were utilized including the following: complexes containing adult cTnT3 and cTnI (TnT3cIC), Tn containing cTnT1 and cTnI (TnT1cIC), Tn containing cTnT4 and cTnI (TnT4cIC), Tn containing cTnT1 and ssTnI (TnT1ssIC), Tn containing cTnT3 and ssTnI (TnT3ssIC), and Tn containing cTnT4 and ssTnI (TnT4ssIC). All Tn complexes contained cTnC. The ATPase reaction was initiated with the addition of ATP and stopped after 20 min with trichloroacetic acid (4% final concentration). After sedimenting the precipitate, the inorganic phosphate concentration in the supernatant was determined according to the method of Fiske and SubbaRow (16Fiske C.H. SubbaRow Y. J. Biol. Chem. 1925; 66: 375-400Abstract Full Text PDF Google Scholar). The ATPase rates were measured at 30 °C by single time points that were predetermined to be linear with time.Determination of the Ca2+Dependence of Force Development, Fiber Preparation—All skinned muscle experiments were performed with glycerinated porcine papillary muscle preparations. Hearts from newly slaughtered pigs were obtained from a nearby slaughterhouse. Strips of muscle, 3–5 millimeters (mm) in diameter and ∼5 mm in length were dissected from the papillary muscle of the left ventricle and skinned for 24 h in a 1% Triton X-100 (by volume) containing 49.5% pCa 8.0 relaxing solution (15Szczesna D. Zhang R. Zhao J. Jones M. Guzman G. Potter J.D. J. Biol. Chem. 2000; 275: 624-630Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Fibers were then transferred to a similar replacement solution without Triton X-100. The skinned cardiac muscle (∼120 μm in diameter) was mounted using stainless steel clips to a force transducer and immersed in a relaxation solution (pCa 8.0) containing 10–8m [Ca2+], 5 mm [Mg2+], 7 mm EGTA, 20 mm imidazole, pH 7.0, 5 mm [Mg2+-ATP], 15 mm creatine phosphate, and 20 units/ml phosphocreatine kinase and between 76 and 92 mm potassium propionate to achieve a constant ionic strength of 150 mm in all solutions (17Guth K. Potter J.D. J. Biol. Chem. 1987; 262: 13627-13635Abstract Full Text PDF PubMed Google Scholar). The contraction solution (pCa 4.0) was the same composition as the pCa 8.0 solution except that the Ca2+ concentration was 10–4m and was used to measure the initial force. All fiber studies were carried out at room temperature (22 °C). To determine the dependence of force development, the contraction of skinned cardiac muscle was tested in solutions containing intermediate concentrations of Ca2+. The various pCa solutions were prepared by using the room temperature (22 °C) binding constants of Fabiato and Fabiato (18Fabiato A. Fabiato F. J. Physiol. (Lond.). 1978; 276: 233-255Crossref Scopus (827) Google Scholar).Treatment of the skinned fibers with 0.8 mg/ml of cTnT (isoforms 1, 3, or 4) for 2 h at room temperature (with fresh cTnT solution changed after 1 h of incubation) resulted in a loss of Ca2+ dependence of force, and the fibers became unregulated. The protein concentration of cTnT was adjusted to 0.8 mg/ml to obtain maximal displacement of Tn subunits and loss of Ca2+-regulated force. When these fibers were incubated with a preformed cTnI·cTnC or ssTnI·cTnC complex (30 μm), in relaxing solution (pCa 8), the fibers underwent a gradual relaxation as the Tn activity was reconstituted (4Gomes A.V. Guzman G. Zhao J. Potter J.D. J. Biol. Chem. 2002; 277: 35341-35349Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 15Szczesna D. Zhang R. Zhao J. Jones M. Guzman G. Potter J.D. J. Biol. Chem. 2000; 275: 624-630Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Once fully relaxed (after1hof incubation with the TnI·cTnC complex), the force became entirely regulated by Ca2+.Determination of Ca2+Dissociation Rates—Ca2+ dissociation rates (koff) were measured using an Applied Photophysics Ltd. (Leatherhead, UK) model SX.18 MV stopped-flow instrument with a dead time of 1.4 ms at 15 °C. The Tn samples were excited using a 150-watt xenon arc source. koff was determined using the fluorescent Ca2+ chelator Quin-2 (19Tikunova S.B. Rall J.A. Davis J.P. Biochemistry. 2002; 41: 6697-6705Crossref PubMed Scopus (55) Google Scholar, 20Davis J.P. Rall J.A. Alionte C. Tikunova S.B. J. Biol. Chem. 2004; 279: 17348-17360Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Quin-2 was excited at 330 nm with its emission monitored through a 510-nm broad bandpass interference filter (Oriel (Stratford, CT)). Each koff represents the mean of at least 21 traces fit with a single exponential, or a double exponential (variance <1.0 × 10–4) over multiple time windows leading to similar results to account for the fact that Quin-2 reports the rates of Ca2+ dissociation from both the N-(site II) and C-domain (sites III and IV) of cTnC in the TnI·cTnC and Tn complexes. The buffer used in all stopped-flow experiments was 10 mm MOPS, 195 mm KCl, 1 mm DTT, 1 mm Mg2+ at pH 7.0. The changes in Quin-2 fluorescence as it binds Ca2+ that dissociates from cTnC in isolation and the various complexes were converted to moles of Ca2+ dissociating from cTnC by mixing increasing concentrations of Ca2+ (0, 15, and 30 μm) with Quin-2 (21Johnson J.D. Snyder C. Walsh M. Flynn M. J. Biol. Chem. 1996; 271: 761-767Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Quin-2 fluorescence increased linearly as a function of increasing [Ca2+], allowing for a conversion of the change in Quin-2 fluorescence to the number of moles of Ca2+ dissociating per mol of cTnC. Calibration curves were performed at the end of an experiment using the same Quin-2 solutions and experimental conditions as used in the experiments. The amplitude of the change in Quin-2 fluorescence was extrapolated from the exponential fits of the data.No additional Ca2+ was added to the samples because there was enough contaminating Ca2+ in the buffers to nearly saturate the complexes. Addition of 15 or 30 μm Ca2+ to the various TnI·cTnC and Tn complexes prior to Quin-2 Ca2+ dissociation led to similar cTnC N-domain Ca2+ dissociation rates and stoichiometries. However, the addition of extra Ca2+ to the reactions abolished the ability of Quin-2 to remove Ca2+ from the C-terminal sites of cTnC in the various complexes.Solubility of cTnT Isoforms—cTnT1, cTnT3, or cTnT4 (1 ml each) at various concentrations (0.4, 0.25, and 0.1 mg/ml) were dialyzed against 50 mm Tris-HCl, pH 7.0, containing 1 mm DTT and 1 m KCl. The cTnT isoforms were then successively dialyzed against the same buffer containing lower concentrations of KCl (0.7, 0.5, 0.4, 0.3, 0.2, and then 0.1 m KCl). After dialysis in buffer containing 0.1 m KCl, the cTnT isoforms were centrifuged at 15,000 rpm for 25 min at 4 °C, and the supernatant was collected. The total volume of the cTnT isoforms obtained after dialysis as well as the concentration of the cTnT isoforms in the supernatant after centrifugation were determined. The protein concentration was determined with the Coomassie Plus Bradford assay kit (Pierce) using bovine serum albumin as standard.Data Analysis—Values are presented as mean ± S.D. The statistical significance of the differences between the mean values was analyzed by the Student's t test.RESULTSActomyosin ATPase Studies on Reconstituted Troponin Complexes Containing Different Troponin T and Troponin I Isoforms—The maximal ATPase activity for Tn complexes containing the different cTnT and cTnI isoforms in the presence of Ca2+ was similar for all the isoforms in the presence of cTnI but different in the presence of ssTnI (Fig. 2A). The myosin ATPase activity in the absence of Tn was considered to be 100% activity (0% inhibition). The ATPase activity results are presented as a bar chart so that the different cTnT and TnI combinations can be directly compared. In all experiments the amount of Tn required for maximal ATPase activation was 1 μm, which is consistent with a ratio of Tn:Tm of 1:1. The Tn complex containing cTnT3 and cTnI (TnT3cIC) showed slightly lower (but not statistically significant) maximal ATPase activity than the other Tn complexes (TnT1cIC or TnT4cIC), similar to what has been reported previously (4Gomes A.V. Guzman G. Zhao J. Potter J.D. J. Biol. Chem. 2002; 277: 35341-35349Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) (Fig. 2A). However, a Tn complex containing cTnT1 and ssTnI (TnT1ssIC) showed a significantly higher maximal ATPase activity (175 ± 5.8%) than both TnT3ssIC (157 ± 3%) and TnT4ssIC (144 ± 5%). The maximal activity for TnT4ssIC was also significantly lower than the maximal activity of TnT4cIC (Fig. 2A).Fig. 2Effect of the cTnT isoforms on the activation (+Ca2+) and inhibition (–Ca2+) of the actin-Tm-activated myosin-ATPase activity.A, activation of ATPase activity. The protein concentrations used in this assay are as follows: 3.5 μm actin, 1 μm Tm, 1 μm Tn, and 0.6 μm myosin. Each data point represents the average of 4–6 separate experiments each performed in triplicate and is expressed as mean ± S.D. The myosin ATPase activity that occurs in the absence of Tn is considered 100% ATPase activity. TnT1ssIC, cTnT1·ssTnI·cTnC complex; TnT3ssIC, cTnT3·ssTnI·cTnC; TnT4ssIC, cTnT4·ssTnI·cTnC complex; TnT1cIC, cTnT1·cTnI·cTnC; TnT3cIC, cTnT3·cTnI·cTnC complex; TnT4cIC, cTnT4·cTnI·cTnC complex. *, p < 0.01; **, p < 0.001. B, inhibition of the ATPase activity. The protein concentrations used in this assay are as follows: 5 μm actin, 1 μm Tm, 2 μm Tn, and 0.6 μm myosin. The myosin ATPase activity that occurs in the absence of Tn is considered 100% ATPase activity. Each data point represents the average of 4–6 separate experiments each performed in triplicate and is expressed as mean ± S.D. TnT1ssIC, cTnT1·ssTnI·cTnC complex; TnT3ssIC, cTnT3·ssTnI·cTnC; TnT4ssIC, cTnT4·ssTnI·cTnC complex; TnT1cIC, cTnT1·cTnI·cTnC; TnT3cIC, cTnT3·cTnI·cTnC complex; and TnT4cIC, cTnT4·cTnI·cTnC complex. The inhibition of ATPase activity by TnT1ssIC and TnT4ssIC are significantly different from the inhibition of ATPase activity by TnT3ssIC. *, p < 0.05; **, p < 0.001.View Large Image Figure ViewerDownload (PPT)The ability of cTnT isoforms to inhibit actin-Tm-activated myosin ATPase activity in reconstituted thin filaments was also examined to determine whether the inhibitory activity of the Tn complexes containing ssTnI was affected by the deletions in the N-terminal region of cTnT. Although TnT3cIC and TnT4cIC were able to inhibit ATPase activity nearly fully in the presence of EGTA (∼92% at 2 μm TnT3cIC concentration), TnT1cIC was less effective at inhibiting actomyosin ATPase activity (∼ 80% at 2 μm Tn concentration), similar to what has been reported previously (Fig. 2B) (4Gomes A.V. Guzman G. Zhao J. Potter J.D. J. Biol. Chem. 2002; 277: 35341-35349Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). However, in the presence of ssTnI, TnT4ssIC (47 ± 3% ATPase inhibition) was unable to inhibit the actin-Tm-activated myosin ATPase activity as well as TnT3ssIC (66 ± 1%). Complexes containing cTnI were all better at inhibiting ATPase activity than complexes containing ssTnI (Fig. 2B). cTnT4 had similar inhibitory properties to cTnT1 in the presence of ssTnI.Actomyosin ATPase assays were also carried out with equal amounts (50:50%) of different Tn complexes (Fig. 3, A and B). All assays in the presence of Ca2+ were carried out with a total Tn concentration of 1 μm. None of the cTnT isoforms showed a significant dominance over the other isoforms in the presence of ssTnI in affecting the maximal actomyosin ATPase activity (Fig. 3A). In the absence of Ca2+, 50:50% ratio of different Tn complexes (Fig. 3B) showed that cTnI was dominant over ssTnI for its ability to inhibit ATPase activity. 50:50% ratios of TnT1ssIC:TnT3ssIC showed a small preference for TnT1 over TnT3 with respect to the inhibition of ATPase activity (Fig. 3B). The results shown in Fig. 3B were all done at a total Tn concentration of 2 μm. However, similar assays done at 1 μm gave similar results (data not shown).Fig. 3Effect of the cTnT isoform mixtures on the activation (+Ca2+) or inhibition (–Ca2+) of the actin-Tm-activated myosin-ATPase activity. Each data point represents the average of 4–6 separate experiments each performed in triplicate and is expressed as mean ± S.D. TnT1ssIC, cTnT1·ssTnI·cTnC complex; TnT3ssIC, cTnT3·ssTnI·cTnC; TnT4ssIC, cTnT4·ssTnI·cTnC complex; TnT1cIC, cTnT1·cTnI·cTnC. A, TnT1:TnT3, 50% TnT1ssIC, 50% TnT3ssIC; TnT1: TnT4, 50% TnT1ssIC, 50% TnT4ssIC; TnT3:TnT4, 50% TnT3ssIC, 50% TnT4ssIC. Assays were carried out with a total Tn concentration of 1 μm. B, TnT1cIC and TnT3cIC, 50% TnT1cIC and 50% TnT3cIC; TnT1ssIC:TnT3ssIC, 50% TnT1ssIC, 50% TnT3ssIC. Assays were carried out with a total Tn concentration of 2 μm.*, p < 0.05; **, p < 0.001View Large Image Figure ViewerDownload (PPT)Force Development and the Ca2+Dependence of Force Development—When the adult isoform of cTnT (cTnT3) was used to displace the native porcine cardiac Tn complex, the cTnI·cTnC reconstituted fibers were less sensitive to Ca2+ (ΔpCa50 ∼ –0.15) than intact fibers similar to what has been reported previously (data not shown) (4Gomes A.V. Guzman G. Zhao J. Potter J.D. J. Biol. Chem. 2002; 277: 35341-35349Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). This difference is presumably due to the different Ca2+ responses of the endogenous porcine and the human Tn complexes reconstituted into skinned muscle fibers as reported previously (4Gomes A.V. Guzman G. Zhao J. Potter J.D. J. Biol. Chem. 2002; 277: 35341-35349Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 15Szczesna D. Zhang R. Zhao J. Jones M. Guzman G. Potter J.D. J. Biol. Chem. 2000; 275: 624-630Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Table I summarizes the pCa50 values of the force-pCa relationship and the Hill coefficients (nH) for the fibers treated with different cTnT and TnI isoforms.Table IEffect of cTnT isoforms on the Ca2+ sensitivity of force development (pCa50) and the Hill coefficient (nH) in skinned porcine cardiac muscle fibers reconstituted with slow skeletal troponin IcTnT Isoform utilized in the fibersTnI IsoformpCa50Hill coefficientRelative forceNumber of experimentsnH%ncTnT1cTnI5.56 ± 0.03aIndicates that the pCa50 values for the cTnT1 isoform is significantly different from cTnT3 and cTnT4 in the presence of cTnI (p < 0.05).1.71 ± 0.09cIndicates that the nH values for the cTnT1 isoform is significantly different from cTnT4 in the presence of cTnI (p < 0.05).72.2 ± 2.64cTnT3cTnI5.44 ± 0.021.90 ± 0.0966.5 ± 3.04cTnT4cTnI5.39 ± 0.022.00 ± 0.12dIndicates that the nH values for the cTnT4 isoform in the presence of cTnI is significantly different from cTnT4 in the presence of ssTnI (p < 0.05).67.1 ± 2.73cTnT1ssTnI5.73 ± 0.03bIndicates that the pCa50 values for the cTnT1 isoform is significantly different from cTnT3 and cTnT4 in the presence of ssTnI (p < 0.05).1.44 ± 0.1491.2 ± 2.94cTnT3ssTnI5.62 ± 0.021.69 ± 0.1175.7 ± 2.54cTnT4ssTnI5.56 ± 0.021.56 ± 0.0865.9 ± 3.03a Indicates that the pCa50 values for the cTnT1 isoform is significantly different from cTnT3 and cTnT4 in the presence of cTnI (p < 0.05).b Indicates that the pCa50 values for the cTnT1 isoform is significantly different from cTnT3 and cTnT4 in the presence" @default.
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• W2139834186 date "2004-11-01" @default.
• W2139834186 modified "2023-10-15" @default.
• W2139834186 title "Cardiac Troponin T Isoforms Affect the Ca2+ Sensitivity of Force Development in the Presence of Slow Skeletal Troponin I" @default.
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• W2139834186 doi "https://doi.org/10.1074/jbc.m407340200" @default.
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