Functional Analysis of a Troponin I (R145G) Mutation Associated with Familial Hypertrophic Cardiomyopathy
2002; Elsevier BV; Volume: 277; Issue: 14 Linguagem: Inglês
10.1074/jbc.m108912200
ISSN1083-351X
AutoresRosalyn Lang, Aldrin V. Gomes, Jiaju Zhao, Todd Miller, James D. Potter, Philippe R. Housmans,
Tópico(s)Cardiovascular Effects of Exercise
ResumoFamilial hypertrophic cardiomyopathy has been associated with several mutations in the gene encoding human cardiac troponin I (HCTnI). A missense mutation in the inhibitory region of TnI replaces an arginine residue at position 145 with a glycine and cosegregates with the disease. Results from several assays indicate that the inhibitory function of HCTnIR145G is significantly reduced. When HCTnIR145G was incorporated into whole troponin, TnR145G(HCTnT·HCTnIR145G·HCTnC), only partial inhibition of the actin-tropomyosin-myosin ATPase activity was observed in the absence of Ca2+ compared with wild type Tn (HCTnT·HCTnI·HCTnC). Maximal activation of actin-tropomyosin-myosin ATPase in the presence of Ca2+ was also decreased in TnR145G when compared with Tn. Using skinned cardiac muscle fibers, we determined that in comparison with the wild type complex 1) the complex containing HCTnIR145G only inhibited 84% of Ca2+-unregulated force, 2) the recovery of Ca2+-activated force was decreased, and 3) there was a significant increase in the Ca2+ sensitivity of force development. Computer modeling of troponin C and I variables predicts that the primary defect in TnI caused by these mutations would lead to diastolic dysfunction. These results suggest that severe diastolic dysfunction and somewhat decreased contractility would be prominent clinical features and that hypertrophy could arise as a compensatory mechanism. Familial hypertrophic cardiomyopathy has been associated with several mutations in the gene encoding human cardiac troponin I (HCTnI). A missense mutation in the inhibitory region of TnI replaces an arginine residue at position 145 with a glycine and cosegregates with the disease. Results from several assays indicate that the inhibitory function of HCTnIR145G is significantly reduced. When HCTnIR145G was incorporated into whole troponin, TnR145G(HCTnT·HCTnIR145G·HCTnC), only partial inhibition of the actin-tropomyosin-myosin ATPase activity was observed in the absence of Ca2+ compared with wild type Tn (HCTnT·HCTnI·HCTnC). Maximal activation of actin-tropomyosin-myosin ATPase in the presence of Ca2+ was also decreased in TnR145G when compared with Tn. Using skinned cardiac muscle fibers, we determined that in comparison with the wild type complex 1) the complex containing HCTnIR145G only inhibited 84% of Ca2+-unregulated force, 2) the recovery of Ca2+-activated force was decreased, and 3) there was a significant increase in the Ca2+ sensitivity of force development. Computer modeling of troponin C and I variables predicts that the primary defect in TnI caused by these mutations would lead to diastolic dysfunction. These results suggest that severe diastolic dysfunction and somewhat decreased contractility would be prominent clinical features and that hypertrophy could arise as a compensatory mechanism. Familial hypertrophic cardiomyopathy (FHC) 1The abbreviations used are: FHCfamilial hypertrophic cardiomyopathyTntroponinCTncardiac TnHCTnhuman CTnTmtropomyosinMOPS4-morpholinepropanesulfonic acidWTwild type has been linked to mutations in genes of nine different sarcomeric proteins. These mutations have been found in the genes for α-myosin heavy chain (1.Geisterfer-Lowrance A.A. Kass S. Tanigawa G. Vosberg H.P. McKenna W. Seidman C.E. Seidman J.G. Cell. 1990; 62: 999-1006Abstract Full Text PDF PubMed Scopus (1054) Google Scholar), cardiac myosin essential light chain and cardiac myosin regulatory light chain (2.Poetter K. Jiang H. Hassanzadeh S. Master S.R. Chang A. Dalakas M.C. Rayment I. Sellers J.R. Fananapazir L. Epstein N.D. Nat. 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In general, patients with FHC demonstrate an increase in heart muscle mass and sometimes an irregular echocardiogram (10.Marian A.J. Roberts R. J. Cardiovasc. Electrophysiol. 1998; 9: 88-99Crossref PubMed Scopus (93) Google Scholar). familial hypertrophic cardiomyopathy troponin cardiac Tn human CTn tropomyosin 4-morpholinepropanesulfonic acid wild type Kimura et al. (6.Kimura A. Harada H. Park J.E. Nishi H. Satoh M. Takahashi M. Hiroi S. Sasaoka T. Ohbuchi N. Nakamura T. Koyanagi T. Hwang T.H. Choo J.A. Chung K.S. Hasegawa A. Nagai R. Okazaki O. Nakamura H. Matsuzaki M. Sakamoto T. Toshima H. Koga Y. Imaizumi T. Sasazuki T. Nat. Genet. 1997; 16: 379-382Crossref PubMed Scopus (472) Google Scholar) reported five missense mutations in TnI, R145G, R145Q, R162W, G203S, and K206Q, that cosegregate with FHC (Fig. 1). Three other TnI FHC mutants (S199N, Lys-183 deletion, and an exon 8 deletion mutant encompassing the stop codon of the cardiac TnI gene) have recently been discovered (11.Morner S. Richard P. Kazzam E. Hainque B. Schwartz K. Waldenstrom A. J. Mol. Cell. Cardiol. 2000; 32: 521-525Abstract Full Text PDF PubMed Scopus (30) Google Scholar, 12.Kokado H. Shimizu M. Yoshio H. Ino H. Okeie K. Emoto Y. Matsuyama T. Yamaguchi M. Yasuda T. Fujino N. Ito H. Mabuchi H. Circulation. 2000; 102: 663-669Crossref PubMed Scopus (85) Google Scholar). Functionally TnI is the inhibitory subunit of the troponin (Tn) complex that controls the interaction between actin and myosin in a Ca2+-dependent manner (13.Greaser M. Gergely J. J. Biol. Chem. 1971; 246: 4226-4233Abstract Full Text PDF PubMed Google Scholar, 14.Sheng Z. Pan B. Miller T.E. Potter J.D. J. Biol. Chem. 1992; 267: 25407-25413Abstract Full Text PDF PubMed Google Scholar, 15.Zot A.S. Potter J.D. Annu. Rev. Biophys. Biophys. Chem. 1987; 16: 535-539Crossref PubMed Scopus (447) Google Scholar). Studies using proteolytic fragments of fast skeletal TnI identified the central TnI sequence (residues 96–116) as being responsible for its inhibitory activity. Residues 104–115 of fast skeletal TnI (comparable to residues 136–147 in cardiac TnI) formed the minimum sequence necessary for inhibition of muscle contraction (16.Talbot J.A. Hodges R.S. J. Biol. Chem. 1981; 256: 12374-12378Abstract Full Text PDF PubMed Google Scholar, 17.Talbot J.A. Hodges R.S. J. Biol. Chem. 1981; 256: 2798-2802Abstract Full Text PDF PubMed Google Scholar, 18.Van Eyk J.E. Hodges R.S. Methods. 1993; 5: 264-280Crossref Scopus (8) Google Scholar, 19.Van Eyk J.E. Hodges R.S. J. Biol. Chem. 1988; 263: 1726-1732Abstract Full Text PDF PubMed Google Scholar). Two of these mutations occur within the inhibitory region of TnI at a highly conserved amino acid residue (R145G and R145Q). The HCTnIR145G mutation has been investigated previously by Elliot et al. (20.Elliott K. Watkins H. Redwood C.S. J. Biol. Chem. 2000; 275: 22069-22074Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) and shown to reduce the inhibition of the actin-Tm-activated myosin ATPase. Additional biochemical studies of the HCTnIR145G mutation and other FHC mutations are necessary to understand the mechanism by which this mutation and other hypertrophic cardiomyopathy mutations cause cardiac hypertrophy and sudden death. Currently the mechanism by which this mutation and other TnI FHC mutations cause cardiac hypertrophy and sudden death is uncertain but may involve primarily defects in relaxation (21.Hernandez O.M. Housmans P.R. Potter J.D. J. Appl. Physiol. 2001; 90: 1125-1136Crossref PubMed Scopus (63) Google Scholar, 22.Knollmann B.C. Potter J.D. Trends Cardiovasc. Med. 2001; 11: 206-212Crossref PubMed Scopus (54) Google Scholar). In the present investigation we have determined how this arginine to glycine change within the inhibitory region of TnI alters the inhibitory function and how this mutation may cause deviations in the normal mechanisms of contraction as an explanation for its association with FHC. This article is the first report of the R145G mutation impairing force development, reducing maximal force, and reducing muscle relaxation. This is the first report of the R145G mutation being used in skinned cardiac fibers and to show that R145G impairs force development and relaxation. Mathematical simulations of intact cardiac fibers, in which the Ca2+ affinity of troponin C and the effectiveness of troponin I as an inhibitor were altered, predict a lower contractility and an increased resting tension in HCTnIR145G myocardium compared with the normal myocardium. These intrinsic contractile changes will likely result in diastolic dysfunction in vivo. The HCTnI mutants (R145G and A86T,R145G) were formed by overlapping PCR using HCTnI cDNA previously cloned in our laboratory from human cardiac tissue (23.Zhang R. Zhao J. Mandveno A. Potter J.D. Circ. Res. 1995; 76: 1028-1035Crossref PubMed Scopus (274) Google Scholar). The sequence of the TnI mutants was verified by sequencing prior to expression and purification. HCTnI, HCTnIR145G, and HCTnIA86T,R145G were purified via conventional methods. Briefly, crude bacterial supernatants were purified by column chromatography on an S-Sepharose column at 4 °C and eluted with a linear KCl gradient of 0–0.5m in a Tris-HCl buffer containing 6 m urea. Semipure HCTnI and HCTnI mutants were dialyzed against a solution containing 50 mm Tris-HCl, pH 7.5, 1 m KCl, 1m urea, 1 mm dithiothreitol, and 2 mm CaCl2 and loaded onto an affinity column having covalently bound HCTnC. Pure HCTnI and HCTnI mutants were eluted with a gradient of 0–3 mm EDTA and 1–6 murea. The purity of the TnI proteins was determined by SDS-PAGE (Fig. 1B). Formation of the human cardiac troponin complexes containing recombinant TnT, TnC, and TnI was carried out as recently described by Szczesna et al. (24.Szczesna 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 we do not routinely analyze reconstituted Tns formed by our method by chromatography, gel filtration of these Tn complexes showed that this reconstitution method resulted in a single species. Porcine cardiac myosin, rabbit skeletal F-actin, porcine cardiac tropomyosin, and recombinant human cardiac TnC were prepared as described previously (25.Potter J.D. Methods Enzymol. 1982; 85: 241-263Crossref PubMed Scopus (298) 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, 0.1 mmdithiothreitol, 10 mm MOPS, pH 7. The ATPase activation assay was carried out in the same 1-ml reaction mixture with 1 mm EDTA replaced with 0.5 m CaCl2. F-actin (3.5 μm), myosin (0.6 μm), and Tm (1 μm) were homogenized (24.Szczesna 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) and added to the reaction tube after the addition of buffer and either wild type human cardiac Tn (WTHCTn), human cardiac Tn containing TnIR145G(HCTnR145G), or human cardiac Tn containing TnIA86T,R145G (HCTnA86T,R145G) to the assay tube. The ATPase reaction was initiated with the addition of ATP and stopped after 20 min with 50% trichloroacetic acid. After sedimenting the precipitate, the inorganic phosphate concentration in the supernatant was determined according to the method of Fiske and Subbarow (26.Fiske C.H. Subbarow Y. J. Biol. Chem. 1925; 66: 375Abstract Full Text PDF Google Scholar). The ATPase rates, measured by a single time point, were predetermined to be linear with time. Cardiac skinned muscle fibers were prepared following a common laboratory procedure published by Zhang et al. (23.Zhang R. Zhao J. Mandveno A. Potter J.D. Circ. Res. 1995; 76: 1028-1035Crossref PubMed Scopus (274) Google Scholar). Freshly isolated porcine hearts were incubated in an O2-saturated solution containing 140 mm NaCl, 4 mm KCl, 1.8 mm CaCl2, 1 mm MgCl2, 1.8 mmNaHPO4, 5.5 mm glucose, and 5 mmHEPES, pH 7.4. The hearts were obtained within 1 h of slaughter from a local slaughterhouse near the University of Miami. Cardiac muscle bundles were dissected from the left ventricle of the porcine hearts and were chemically skinned by incubating with 50% glycerol and 1% Triton X-100 in the relaxing solution (pCa 8.0) containing 10−8m Ca2+, 5 mm Mg2+, 7 mm EGTA, 20 mm MgATP, 20 mm creatine phosphate, and 15 units/ml creatine phosphokinase, pH 7.0, at an ionic strength of 150 mm at 4 °C for 24 h. These skinned muscle preparations were dissected into small bundles (1–2 cm in length, 2–3 mm in diameter) and were stored at −20 °C in the same solution without Triton X-100 before use. The skinned fiber preparation was mounted with stainless steel clips on a force transducer and was immersed in the contracting solution to measure initial force before treatment. The contraction solution (pCa 4) had the same composition as the relaxation buffer except for the increased Ca2+ concentration (10−4m). To determine the Ca2+dependence of force development, the contraction of the skinned fibers was tested in solutions of intermediate concentrations of Ca2+. The Ca2+ dependence of force was determined before and after performing the displacement and reconstitution protocols that are described below. To remove the endogenous Tn complex, the TnT displacement method was used (24.Szczesna 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, 27.Hatakenaka M. Ohtsuki I. FEBS Lett. 1992; 205: 985-993Google Scholar). The cardiac fiber was incubated with HCTnT (0.8 mg/ml) for a total of 2.5 h with an intermediate buffer change containing fresh HCTnT. After displacement of the endogenous complex by HCTnT, the level of unregulated force development was observed by measuring the level of force reached by skinned fibers in both the pCa 8 solution and the pCa 4 solution. To determine the Ca2+dependence of force development, the contraction of skinned fibers was tested in solutions with intermediate concentrations of Ca2+ (from pCa 8 to pCa 4). The Ca2+ dependence was determined before and after treatment of the skinned fibers with displacement and reconstitution solutions. The Ca2+ dependence data were fit to the Hill equation with SIGMAPLOT (Jandel Scientific): relative force (%) = [Ca2+]n/([Ca2+]n+ [Ca50]n), where p K is the midpoint (pCa50) and n is the Hill coefficient. Control experiments carried out on porcine fibers treated the same way as described above but without any protein showed that the average rundown for these fibers was ∼12–18%. Taking this average rundown of the fibers into account the average recovered force for these skinned fiber studies is ∼70% of the original force before treatment with TnT and reconstitution with TnI-TnC. The average force at the end of these experiments is ∼60% of the initial force. All data are presented as mean ± S.D. CD spectra of the HCTnI and two mutants were recorded on a Jasco J-720 spectropolarimeter using a cell pathlength of 0.1 cm at ambient temperature (20 °C) in a 10 mm sodium phosphate, pH 7.0, 500 mm NaCl solution. Spectra were recorded at 200–250 nm with a bandwidth of 1 nm at a speed of 50 nm/min and a resolution of 0.2 nm. Analysis and processing of data was done using the Jasco system software (Windows Standard Analysis, version 1.20). Ten scans were averaged, base lines were subtracted, and no numerical smoothing was applied. Mean residue ellipticity ([θ]MRE, in degrees·cm2/dmol) for the spectra were calculated (using the Jasco system software) using the following equation: [θ]MRE = [θ]/(10·Cr·l), where [θ] is the measured ellipticity in millidegrees, Cr is the mean residue molar concentration, and l is the path length in cm. The α-helical content for each protein was calculated using the standard equation for [θ] at 222 nm (28.Chen Y.H. Yang J.T. Biochem. Biophys. Res. Commun. 1971; 44: 1285-1291Crossref PubMed Scopus (328) Google Scholar): [θ]222 = −30,300fH − 2,340, where fH is the fraction of α-helical content (fH × 100, expressed in %). Spectra are presented as the mean residue ellipticity. To assess the impact of changes incurred in force regulation in cardiac skinned fibers by HCTnIR145G, we used a quantitative computer model that integrates Ca2+ binding to various buffers in cardiac cells to predict the amplitude and time course of the intracellular Ca2+ transient, of Ca2+ bound to each of various Ca2+ buffers (TnC, calmodulin, and sarcoplasmic reticulum uptake), and of force. This method quantifies buffering of intracellular Ca2+, sarcoplasmic reticulum Ca2+ release and uptake, and free [Ca2+]i based on an initially assumed intracellular free Ca2+ transient and published rate constants (29.Fabiato A. Am. J. Physiol. 1983; 245: C1-C14Crossref PubMed Google Scholar, 30.Blinks J.R. Endoh M. Circulation. 1986; 73: III85-III98PubMed Google Scholar). The model is similar to that of Robertson et al. (31.Robertson S.P. Johnson J.D. Potter J.D. Biophys. J. 1981; 34: 559-569Abstract Full Text PDF PubMed Scopus (286) Google Scholar) and in addition incorporates the following processes: (a) a variation of the Ca2+ affinity of TnC with developed force (koff =koff.rest e−gain × relative force) (33.Housmans P.R. Wanek L.A. Carton E.G. Bartunek A.E. Anesthesiology. 2000; 93: 189-201Crossref PubMed Scopus (33) Google Scholar, 34.Landesberg A. Sideman S. Am. J. Physiol. 1994; 266: H1260-H1271PubMed Google Scholar), (b) a modulation of TnI effectiveness as an inhibitor dependent primarily on the concentration of the Ca2+·TnC complex (TnIeffect = [Tn] × (1 − TiAmp × e−TiPower × [CaT]), where TiAmp and TiPower are amplitude and gain variables of the effectiveness of TnI to inhibit actomyosin interactions, and (c) a two-state cross-bridge model (32.Mikane T. Araki J. Kohno K. Nakayama Y. Suzuki S. Shimizu J. Matsubara H. Hirakawa M. Takaki M. Suga H. Am. J. Physiol. 1997; 273: H2891-H2898PubMed Google Scholar). Extensive details of this quantitative computer model can be found in the methods and appendix sections of Miller et al. (35.Miller T. Szczesna D. Housmans P.R. Zhao J. de Freitas F. Gomes A.V. Culbreath L. McCue J. Wang Y. Xu Y. Kerrick W.G. Potter J.D. J. Biol. Chem. 2001; 276: 3743-3755Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) along with the additional features mentioned above. The terms used in this computer model are briefly described below. The rate of release of Ca2+ from TnC is slowed by the presence of cross-bridges. koff(TnC·Ca) therefore becomes smaller as cross-bridges form and force develops (34.Landesberg A. Sideman S. Am. J. Physiol. 1994; 266: H1260-H1271PubMed Google Scholar, 37.Zot A.S. Potter J.D. Biochemistry. 1989; 28: 6751-6756Crossref PubMed Scopus (44) Google Scholar, 38.Hofmann P.A. Fuchs F. Am. J. Physiol. 1987; 253: C541-C546Crossref PubMed Google Scholar). The exponential dependence of koff(TnC·Ca) on force is based on experimental observations in cardiac and skeletal muscle (32.Mikane T. Araki J. Kohno K. Nakayama Y. Suzuki S. Shimizu J. Matsubara H. Hirakawa M. Takaki M. Suga H. Am. J. Physiol. 1997; 273: H2891-H2898PubMed Google Scholar, 33.Housmans P.R. Wanek L.A. Carton E.G. Bartunek A.E. Anesthesiology. 2000; 93: 189-201Crossref PubMed Scopus (33) Google Scholar, 34.Landesberg A. Sideman S. Am. J. Physiol. 1994; 266: H1260-H1271PubMed Google Scholar) and is quantified by the equation koff.TnC·Ca = koff.TnC·Ca.rest × e−gain × relative force, where koff.TnC·Ca.restis the off-rate of Ca2+ from TnC in the absence of force development. For the simulations used in this study, gain was set at 8. Fig. 7A illustrates the strong dependence of the dissociation rate of Ca2+ from TnC as a function of force. The model assumes that for each Ca2+·TnC complex, an effective relief of inhibition by TnI occurs according to the following relationship: TnIeff = [Tn] × (1 − TiAmp × e−TiPower × [CaT], where TiAmp and TiPower are amplitude and gain variables of TnI. In control conditions (WT), TiAmp = 1 and TiPower = 10,000. These values were found by multiple iterations in values of TiPower, gain of TnC-Ca2+binding, and koff.TnC·Ca.rest to reproduce the values found for skinned fibers containing HCTnI (WT). Fig. 7B (solid line) illustrates the relationship between [CaT] and effective [TnI]. The latter value was used as a proxy for [CaT] in further calculations of force. For the cross-bridge on and off kinetics, Huxley's 1957 model (36.Huxley A.F. Prog. Biophys. Biophys. Chem. 1957; 7: 255-318Crossref PubMed Google Scholar) (d n/d t =f × (1 − n) − g × n, where n = number of attached cross-bridges) was modified as follows: d[CB]/d t = f × [TnIeff] × ([total CB] − [CB]) − g × [CB], where [CB] is the instantaneous concentration of attached cross-bridges, [total CB] is the total number of cross-bridges (150 μm·kg−1) (32.Mikane T. Araki J. Kohno K. Nakayama Y. Suzuki S. Shimizu J. Matsubara H. Hirakawa M. Takaki M. Suga H. Am. J. Physiol. 1997; 273: H2891-H2898PubMed Google Scholar), f is the attachment (on) rate constant of detached cross-bridges, g is the detachment (off) rate of attached cross-bridges, and TnIeff is the effective TnI concentration permitting actomyosin interactions consequent to Ca2+ occupancy of TnC. We assumed constant values for f = 400,000 mol·kg−1·s−1 and g = 10 s−1. Finally force is displayed as normalized to maximal force that could theoretically be obtained, i.e. as the ratio [CB]/[total CB]. Simulations for reproducing experimental results of skinned fibers were carried out for equilibrium conditions in pCa values of 8–4 in 0.1-pCa steps. Values of koff.TnC·Ca.rest, gain (TnC-Ca2+ binding dependence on force), and TiAmp and TiPower (amplitude and gain of TnI effectiveness as an inhibitor) were varied until a unique combination of values produced pCa-force relations with identical pCa50 and Hill n coefficients as the wild type. The same procedure was carried out to simulate pCa-force relations of the HCTnIR145F mutant for which a unique convergent solution was found. Cross-bridge variables were not changed. Subsequently variables found from simulations in skinned fibers were fed into the simulation programs that generate twitch contractions of intact cardiac fibers. In the initial control conditions, once sarcoplasmic reticulum Ca2+ release was obtained, steady-state control conditions were obtained by repeating the simulation over several successive twitch contractions by setting the initial values of [Ca2+], [CaT], [CaC], and [CB] of a given twitch to their values obtained at the end of the previous twitch (where [CaC] is calmodulin occupied with Ca2+ and [CaT] is troponin occupied with Ca2+). Simulations carried out for a change in one or more rate constants or variables were also allowed to reach steady-state conditions over several twitches. In most instances, steady-state conditions were reached in 3–5 contractions at frequencies corresponding to heart rates of 150 min−1. We simulated force during a twitch that would occur if one or more of the following would be affected by the presence of HCTnIR145G on the thin filament: (a) a decrease in the resting (zero force) off-rate Ca2+ from the Ca2+-specific site of TnC (koff(TnC·Ca)) from a control value of 700 s−1 and/or (b) a decrease in the amplitude (TiAmp) and gain (TiPower) of troponin I to inhibit actomyosin interactions. We chose not to alter cross-bridge kinetic variables as there is no a priori reason to assume that the mutation would directly affect actomyosin kinetic behavior. Based on each possible effect of HCTnIR145G (see "Results") this theoretical analysis predicts muscle contraction and relaxation as would be encountered in vivo and provides for a series of hypotheses that could be tested in the hearts of HCTnIR145Gtransgenic animals. The calculations were programmed in Microsoft Quickbasic 4.0 and run on a personal computer, and the results (saved as ASCII files) were replotted with SigmaPlot 5.0 (SPSS, Inc., Richmond, CA). Fig. 1A is a diagrammatical representation of the primary sequence of human cardiac TnI showing regions known to be involved in binding TnC, TnT, and tropomyosin. All identified FHC TnI mutations are shown by arrows and occur on the COOH-terminal half of TnI. Residue 86 in the NH2-terminal half of TnI is also shown by an arrow and represents a conflict in the first HCTnI sequence published by Vallins et al. (where residue 86 was Thr (41.Vallins W.J. Brand N.J. Dabhade N. Butler-Browne G. Yacoub M.H. Barton P.J. FEBS Lett. 1990; 270: 57-61Crossref PubMed Scopus (127) Google Scholar)) and later HCTnI sequences that coded for Ala at residue 86 (42, 43). Fig. 1B shows an SDS-polyacrylamide gel of purified wild type and mutant TnIs used in these studies. We examined the ability of HCTnIR145G and WTHCTnI to inhibit F-actin-Tm-activated myosin ATPase activity in reconstituted thin filament systems to determine whether the inhibitory activity of the HCTnIR145Gmutant of TnI was affected by the missense mutation within the inhibitory region of the protein. We discovered that while the wild type HCTnI was able to inhibit ATPase activity nearly fully (approximately 90% at 3 μm HCTnI concentration), HCTnIR145G was less effective at inhibiting actomyosin ATPase activity even at higher protein concentrations (<60% at 4 μm HCTnI concentration, Fig. 2). When TnR145G was reconstituted with actin, Tm, and myosin in the presence of EGTA, the ability of the mutant complex to inhibit ATPase activity was significantly less (20–25% lower at Tn concentrations between 1–2 μm) than wild type Tn (Fig. 3A). In the presence of EGTA wild type Tn (2 μm) inhibited the actin-TM-activated myosin ATPase activity by 82 ± 4%. Under the same conditions used for the wild type Tn, HCTnR145G inhibited the actin-TM-activated myosin ATPase activity by 61 ± 4%. The maximum ATPase activity for TnR145G in the presence of Ca2+ was also less (25% lower at Tn concentrations between 1–2 μm) than wild type Tn (Fig. 3B). In these experiments the amount of Tn required for maximal ATPase activation was 1–1.5 μm, which is consistent with a ratio of Tn:Tm of 1:1(which is considered to be the physiological ratio of these components in intact muscle fibers). The amount of Tn required for maximal ATPase inhibition (in the absence of Ca2+) was 1.5–2 μm. We used a well established method in our laboratory (24.Szczesna 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) to displace the endogenous Tn complex from skinned porcine skinned cardiac muscle preparations. In these experiments, after displacing the endogenous porcine Tn complex with HCTnT (0.8 mg/ml) either wild type HCTnI·HCTnC, HCTnIR145G·HCTnC, or HCTnIA86T,R145G·HCTnC complexes were used to reconstitute the HCTnT-replaced skinned fibers. After determining the level of unregulated force in skinned fibers, they were incubated with either wild type or mutant HCTnI·HCTnC complexes in low calcium buffer (pCa 8). This allowed us to determine whether the TnI proteins could fully inhibit Ca2+-unregulated force established after treatment with HCTnT and also to determine whether the proteins were able to fully reconstitute the skinned fibers by forming a functional Tn complex. Wild type HCTnI·CTnC complex resulted in complete inhibition of Ca2+-unregulated force. However, when the skinned fibers were incubated with the HCTnIR145G·HCTnC complex, only 84% inhibition of unregulated force was observed (Figs. 4 and 5B). Full inhibition of unregulated force was not achieved with the complex containing the mutant TnI even after an extended incubation time of 2.5 h (data not shown). The data depicted in the absence of Ca2+ represents the level of force remaining after reconstituting the fibers with the appropriate TnI·TnC complex (Figs. 4 and 5B). This level of force is equivalent to the percentage of Ca2+-unregulated force that was not inhibited by the Tn complex. The fibers reconstituted with TnR145G were able to inhibit only 84% of unregulated force compared with 100% inhibition by wild type Tn. Recovered force is equivalent to the level of force developed in fibers after reconstituting the fibers with the appropriate Tn complex and treating the fiber with pCa 4 solution. Recove
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