Alterations in Thin Filament Regulation Induced by a Human Cardiac Troponin T Mutant That Causes Dilated Cardiomyopathy Are Distinct from Those Induced by Troponin T Mutants That Cause Hypertrophic Cardiomyopathy

We have compared the in vitroregulatory properties of recombinant human cardiac troponin reconstituted using wild type troponin T with troponin containing the ΔLys-210 troponin T mutant that causes dilated cardiomyopathy (DCM) and the R92Q troponin T known to cause hypertrophic cardiomyopathy (HCM). Troponin containing ΔLys-210 troponin T inhibited actin-tropomyosin-activated myosin subfragment-1 ATPase activity to the same extent as wild type at pCa8.5 (>80%) but produced substantially less enhancement of ATPase atpCa4.5. The Ca2+ sensitivity of ATPase activation was increased (ΔpCa50 = +0.2pCa units) and cooperativity of Ca2+ activation was virtually abolished. Equimolar mixtures of wild type and ΔLys-210 troponin T gave a lower Ca2+ sensitivity than with wild type, while maintaining the diminished ATPase activation atpCa4.5 observed with 100% mutant. In contrast, R92Q troponin gave reduced inhibition at pCa8.5 but greater activation than wild type at pCa4.5; Ca2+sensitivity was increased but there was no change in cooperativity.In vitro motility assay of reconstituted thin filaments confirmed the ATPase results and moreover indicated that the predominant effect of the ΔLys-210 mutation was a reduced sliding speed. The functional consequences of this DCM mutation are qualitatively different from the R92Q or any other studied HCM troponin T mutation, suggesting that DCM and HCM may be triggered by distinct primary stimuli. We have compared the in vitroregulatory properties of recombinant human cardiac troponin reconstituted using wild type troponin T with troponin containing the ΔLys-210 troponin T mutant that causes dilated cardiomyopathy (DCM) and the R92Q troponin T known to cause hypertrophic cardiomyopathy (HCM). Troponin containing ΔLys-210 troponin T inhibited actin-tropomyosin-activated myosin subfragment-1 ATPase activity to the same extent as wild type at pCa8.5 (>80%) but produced substantially less enhancement of ATPase atpCa4.5. The Ca2+ sensitivity of ATPase activation was increased (ΔpCa50 = +0.2pCa units) and cooperativity of Ca2+ activation was virtually abolished. Equimolar mixtures of wild type and ΔLys-210 troponin T gave a lower Ca2+ sensitivity than with wild type, while maintaining the diminished ATPase activation atpCa4.5 observed with 100% mutant. In contrast, R92Q troponin gave reduced inhibition at pCa8.5 but greater activation than wild type at pCa4.5; Ca2+sensitivity was increased but there was no change in cooperativity.In vitro motility assay of reconstituted thin filaments confirmed the ATPase results and moreover indicated that the predominant effect of the ΔLys-210 mutation was a reduced sliding speed. The functional consequences of this DCM mutation are qualitatively different from the R92Q or any other studied HCM troponin T mutation, suggesting that DCM and HCM may be triggered by distinct primary stimuli. Dilated cardiomyopathy (DCM) 1The abbreviations used are: DCM, dilated cardiomyopathy; HCM, hypertrophic cardiomyopathy; S-1, subfragment 1; PIPES, 1,4-piperazinediethanesulfonic acid; HMM, heavy meromyosin. 1The abbreviations used are: DCM, dilated cardiomyopathy; HCM, hypertrophic cardiomyopathy; S-1, subfragment 1; PIPES, 1,4-piperazinediethanesulfonic acid; HMM, heavy meromyosin. is defined clinically by cardiac chamber dilatation with reduced contractile performance in the absence of underlying coronary artery disease. The heart appears thin walled and distended and, at the microscopic level, there is moderate myocyte hypertrophy and death, along with replacement fibrosis. Echocardiographic screening of relatives of affected individuals suggests that ∼25–35% of cases are familial (1Keeling P.J. Gang Y. Smith G. Seo H. Bent S.E. Murday V. Caforio A.L. McKenna W.J. Br. Heart J. 1995; 73: 417-421Google Scholar, 2Michels V.V. Moll P.P. Miller F.A. Tajik A.J. Chu J.S. Driscoll D.J. Burnett J.C. Rodeheffer R.J. Chesebro J.H. Tazelaar H.D. N. Engl. J. Med. 1992; 326: 77-82Google Scholar). The disease is frequently inherited with an associated phenotype such as conduction disease, skeletal myopathy, or sensorineural hearing loss. To date, as many as 18 loci that cause DCM as the predominant phenotype have been identified, and in all but two of these, the disease is inherited in an autosomal dominant manner (3Seidman J.G. Seidman C. Cell. 2001; 104: 557-567Google Scholar, 4Schonberger J. Seidman C.E. Am. J. Hum. Genet. 2001; 69: 249-260Google Scholar). For 10 of the loci, the disease genes have been identified. These encode a diverse range of proteins, including components of the sarcomere: actin (ACTC) (5Olson T.M. Michels V.V. Thibodeau S.N. Tai Y.S. Keating M.T. Science. 1998; 280: 750-752Google Scholar); β-myosin heavy chain (MYH7) (6Kamisago M. Sharma S.D. DePalma S.R. Solomon S. Sharma P. McDonough B. Smoot L. Mullen M.P. Woolf P.K. Wigle E.D. Seidman J.G. Seidman C.E. Jarcho J. Shapiro L.R. N. Engl. J. Med. 2000; 343: 1688-1696Google Scholar); titin (TTN) (7Gerull B. Gramlich M. Atherton J. McNabb M. Trombitas K. Sasse-Klaassen S. Seidman J.G. Seidman C. Granzier H. Labeit S. Frenneaux M. Thierfelder L. Nat. Genet. 2002; 30: 201-204Google Scholar); α-tropomyosin (TPM1) (8Olson T.M. Kishimoto N.Y. Whitby F.G. Michels V.V. J. Mol. Cell Cardiol. 2001; 33: 723-732Google Scholar); and cardiac troponin T (TNNT2) (6Kamisago M. Sharma S.D. DePalma S.R. Solomon S. Sharma P. McDonough B. Smoot L. Mullen M.P. Woolf P.K. Wigle E.D. Seidman J.G. Seidman C.E. Jarcho J. Shapiro L.R. N. Engl. J. Med. 2000; 343: 1688-1696Google Scholar). In a recent report, Kamisago et al. (6Kamisago M. Sharma S.D. DePalma S.R. Solomon S. Sharma P. McDonough B. Smoot L. Mullen M.P. Woolf P.K. Wigle E.D. Seidman J.G. Seidman C.E. Jarcho J. Shapiro L.R. N. Engl. J. Med. 2000; 343: 1688-1696Google Scholar) identified two mutations in β-myosin heavy chain and one in cardiac troponin T (the deletion of lysine 210) in kindreds having autosomal dominant dilated cardiomyopathy without conduction disease, skeletal muscle dysfunction, or other accompanying phenotypes. It was noteworthy that affected subjects did not have ventricular hypertrophy, and histology from one subject showed mildly increased interstitial fibrosis without the myocyte and myofibrillar disarray characteristic of hypertrophic cardiomyopathy (HCM). These mutations therefore appear to cause dilated cardiomyopathy directly and induce a phenotype that is distinctly different from HCM. HCM is known to be caused by mutations in at least 10 genes, all but one of which encodes a sarcomeric protein (3Seidman J.G. Seidman C. Cell. 2001; 104: 557-567Google Scholar, 9Blair E. Redwood C. Ashrafian H. Oliveira M. Broxholme J. Kerr B. Salmon A. Ostman-Smith I. Watkins H. Hum. Mol. Genet. 2001; 10: 1215-1220Google Scholar). In contrast to the contractile protein gene mutations that cause DCM, the functional consequences of the HCM mutations have been extensively characterized (reviewed in Refs. 3Seidman J.G. Seidman C. Cell. 2001; 104: 557-567Google Scholar, 10Redwood C.S. Moolman-Smook J.C. Watkins H. Cardiovasc. Res. 1999; 44: 20-36Google Scholar, and 11Hernandez O.M. Housmans P.R. Potter J.D. J. Appl. Physiol. 2001; 90: 1125-1136Google Scholar). Most mutations in sarcomeric proteins have been found to increase maximum shortening speed and/or Ca2+ sensitivity in vitro, which may result in energetic compromise through increased cost of force productionin vivo (9Blair E. Redwood C. Ashrafian H. Oliveira M. Broxholme J. Kerr B. Salmon A. Ostman-Smith I. Watkins H. Hum. Mol. Genet. 2001; 10: 1215-1220Google Scholar, 12Sweeney H.L. Feng H.S. Yang Z. Watkins H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14406-14410Google Scholar, 13Montgomery D.E. Tardiff J.C. Chandra M. J. Physiol. (Lond.). 2001; 536: 583-592Google Scholar, 14Franz W.M. Muller O.J. Katus H.A. Lancet. 2001; 358: 1627-1637Google Scholar, 15Watkins H. Eur. Heart J. 2001; 3 (suppl.): L43-L50Google Scholar). In HCM families, some individuals go on to develop a dilated cardiomyopathy phenotype, presumably through induction of apoptosis (3Seidman J.G. Seidman C. Cell. 2001; 104: 557-567Google Scholar). Thus, one plausible hypothesis to explain how different mutations in the same gene can cause different cardiomyopathies is that DCM mutations produce similar, but more severe, perturbations of contractile protein function, sufficient to result in cell death. Alternatively, the DCM mutations in sarcomeric protein genes could initiate disease through qualitatively different perturbations of contractility (6Kamisago M. Sharma S.D. DePalma S.R. Solomon S. Sharma P. McDonough B. Smoot L. Mullen M.P. Woolf P.K. Wigle E.D. Seidman J.G. Seidman C.E. Jarcho J. Shapiro L.R. N. Engl. J. Med. 2000; 343: 1688-1696Google Scholar, 16Marston S.B. Hodgkinson J.L. J. Muscle Res. Cell Motil. 2001; 22: 1-4Google Scholar). Functional analysis of different mutations within a single gene that produce the divergent phenotypes of HCM and DCM provides a valuable opportunity to investigate the triggers that discriminate between these two disease pathways. In this report, we have focused on the ΔLys-210 troponin T mutant that causes DCM. In common with the HCM troponin T mutations, it is highly likely that this apparently subtle mutation acts in a dominant-negative manner and is incorporated into the thin filament, where it affects normal thin filament function. The deleted amino acid forms one of a stretch of four lysine residues in human cardiac troponin T (amino acids 207–210). These lie within the globular C-terminal T2 domain (residues 188–288) which binds to troponins I and C as well as to tropomyosin (17Perry S.V. J. Muscle Res. Cell Motil. 1998; 19: 575-602Google Scholar) and may therefore affect thin filament function by a variety of mechanisms. We have compared the changes in thin filament function caused by the ΔLys-210 mutation with those caused by a mutation in troponin T that causes HCM. For the latter we have used the R92Q troponin T mutant, which has been extensively characterized in transgenic mouse models (13Montgomery D.E. Tardiff J.C. Chandra M. J. Physiol. (Lond.). 2001; 536: 583-592Google Scholar, 18Oberst L. Zhao G. Park J.T. Brugada R. Michael L.H. Entman M.L. Roberts R. Marian A.J. J. Clin. Invest. 1998; 102: 1498-1505Google Scholar, 19Tardiff J.C. Hewett T.E. Palmer B.M. Olsson C. Factor S.M. Moore R.L. Robbins J. Leinwand L.A. J. Clin. Invest. 1999; 104: 469-481Google Scholar) and myofibrils (20Morimoto S. Yanaga F. Minakami R. Ohtsuki I. Am. J. Physiol. 1998; 275: C200-C207Google Scholar,21Szczesna D. Zhang R. Zhao J. Jones M. Guzman G. Potter J.D. J. Biol. Chem. 2000; 275: 624-630Google Scholar) 2R. Willott and C. Ashley, unpublished data. 2R. Willott and C. Ashley, unpublished data. but has not yet been examined in reconstituted thin filaments in vitro. We found that the ΔLys-210 mutation had distinctive effects upon thin filament function: the maximally activated ATPase activity and filament sliding speed were decreased, and Ca2+ activation became non-cooperative. This pattern of changes was quite unlike the effect of the R92Q HCM mutation (increased ATPase activity and sliding speed and higher Ca2+ sensitivity with unaltered cooperativity) but closely resembled the properties of troponin extracted from end stage failing human hearts studied by the same techniques (22Purcell I.F. Bing W. Marston S.B. Cardiovasc. Res. 1999; 43: 884-891Google Scholar, 23Knott A. Purcell I.F. Marston S.B. J. Mol. Cell. Cardiol. 2002; 34: 469-482Google Scholar). Rabbit skeletal muscle actin, rabbit and human cardiac muscle α-tropomyosin, and subfragment-1 (S-1) derived by chymotryptic digestion of whole rabbit skeletal muscle myosin were prepared as previously described (22Purcell I.F. Bing W. Marston S.B. Cardiovasc. Res. 1999; 43: 884-891Google Scholar, 24Redwood C.S. Lohmann K. Bing W. Esposito G.M. Elliott K. Abdulrazzak H. Knott A. Purcell I. Marston S.B. Watkins H. Circ. Res. 2000; 86: 1146-1152Google Scholar). Recombinant wild type human troponin subunits were overexpressed in BL21(DE3)pLysS Escherichia coli and subsequently purified (24Redwood C.S. Lohmann K. Bing W. Esposito G.M. Elliott K. Abdulrazzak H. Knott A. Purcell I. Marston S.B. Watkins H. Circ. Res. 2000; 86: 1146-1152Google Scholar). pMW172 expression constructs encoding R92Q and ΔLys-210 troponin T were made, respectively, by subcloning from an existing plasmid cytomegalovirus construct (12Sweeney H.L. Feng H.S. Yang Z. Watkins H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14406-14410Google Scholar) and using a two-step PCR protocol for site-directed mutagenesis. Whole troponin complexes were formed using a development of our established protocol (25Elliott K. Watkins H. Redwood C.S. J. Biol. Chem. 2000; 275: 22069-22074Google Scholar). The subunits were mixed in a ratio of 1.5 troponin C:1 troponin I:1 troponin T in 6 m urea, 1m KCl, 10 mm imidazole, 50 μmCaCl2, 1 mm dithiothreitol, 0.01% sodium azide, pH 7.0, and the concentrations of first urea and then KCl were reduced using a stepwise dialysis protocol to 0 and 200 mm,respectively. The mixtures were centrifuged (12,300 ×g, 5 min) to remove insoluble material and intact troponin purified by gel filtration using a Sepharose 200 column. Complexes were then dialyzed into the appropriate assay buffer. The final proportions of individual subunits were measured by scanning densitometry and found to be 1.00:0.95:1.12 (troponin T:troponin I:troponin C;n = 3) for wild type and both mutants. Assays were carried out as previously described using 0.5 μm myosin S-1 and thin filaments reconstituted using either 3.5 μmactin, 1 μm tropomyosin, and 1 μm troponin or 3.5 μm actin, 0.5 μm tropomyosin, and 0.5 μm troponin in 5 mm PIPES, 3.87 mm MgCl2, 1 mm dithiothreitol, pH 7.0, at 37 °C (25Elliott K. Watkins H. Redwood C.S. J. Biol. Chem. 2000; 275: 22069-22074Google Scholar). The free Ca2+ concentration was set using 1 mm EGTA and the appropriate concentration of CaCl2 as previously described (25Elliott K. Watkins H. Redwood C.S. J. Biol. Chem. 2000; 275: 22069-22074Google Scholar). The movement of thin filaments over a bed of immobilized skeletal muscle heavy meromyosin (HMM) was investigated using the in vitro motility assay as we have described (24Redwood C.S. Lohmann K. Bing W. Esposito G.M. Elliott K. Abdulrazzak H. Knott A. Purcell I. Marston S.B. Watkins H. Circ. Res. 2000; 86: 1146-1152Google Scholar, 26Bing W. Fraser I.D.C. Marston S.B. Biochem. J. 1997; 327: 335-340Google Scholar). F-actin was labeled with rhodamine-phalloidin (φ) as described by Kron et al. (27Kron S.J. Toyoshima Y.Y. Uyeda T.Q. Spudich J.A. Methods Enzymol. 1991; 196: 399-416Google Scholar). HMM at 100 μg/ml was infused in buffer A (50 mm KCl, 25 mmimidazole-HCl, pH 7.4, 4 mm MgCl2, 1 mm EDTA, 5 mm dithiothreitol) to provide a coating of immobilized HMM on the coverslip. The surface was blocked by infusing 0.5 mg/ml bovine serum albumin in buffer A, and then reconstituted thin filaments were infused. All experiments were carried out at 28 °C with the following final concentrations of reconstituted thin filament proteins: 10 nm rabbit skeletal actin, 30 nm human cardiac tropomyosin, 0–20 nm reconstituted human cardiac troponin in buffer A plus 0.1 mg/ml glucose oxidase, 0.02 mg/ml catalase, 3 mg/ml glucose, 0.5% methylcellulose, 1 mm MgATP, ± troponin at assay concentration. Both tropomyosin and troponin were titrated to determine saturating concentrations. The movement of actin (φ) tropomyosin filaments over the immobilized skeletal muscle HMM was observed under a Zeiss epifluorescence microscope (×63/1.4 objective). Four 15 s videos were recorded in each cell before any significant photobleaching had occurred. Videos were digitized, and the movement was analyzed to determine fraction of filaments moving and velocity of motile filaments using the automatic tracking program described by Marston et al. (28Marston S.B. Fraser I.D. Bing W. Roper G. J. Muscle Res. Cell Motil. 1996; 17: 497-506Google Scholar). Troponin complexes were reconstituted from recombinant human cardiac troponins I, C, and T using wild type troponin T, ΔLys-210 mutant troponin T, or R92Q mutant troponin T. Thin filaments were assembled using rabbit skeletal actin, rabbit or human α-tropomyosin, and wild type or mutant troponin. The functional properties of wild type and mutant troponin T were compared by assay of thin filament activation of skeletal muscle myosin S-1 ATPase activity and by thein vitro motility assay using skeletal muscle heavy meromyosin. Actin cosedimentation assays carried out as previously described (25Elliott K. Watkins H. Redwood C.S. J. Biol. Chem. 2000; 275: 22069-22074Google Scholar) showed that the binding of the wild type and both mutant troponins to actin-tropomyosin were indistinguishable (data not shown). In ATPase experiments carried out under activating conditions, the addition of wild type troponin increased ATPase activity, reaching a plateau at 207 ± 3% of the ATPase rate obtained using actin-tropomyosin alone, whereas when troponin containing ΔLys-210 troponin T was added the maximum ATPase activation was only 167 ± 6% of the actin-tropomyosin rate. This difference was consistently seen in four different troponin preparations and was highly significant (p <0.001) (Fig.1 A). At pCa 5.4, troponin increased the speed of actin-tropomyosin filament sliding in the in vitro motility assay, reaching a plateau at 10–15 nm troponin (Fig.2 A). As previously observed (24Redwood C.S. Lohmann K. Bing W. Esposito G.M. Elliott K. Abdulrazzak H. Knott A. Purcell I. Marston S.B. Watkins H. Circ. Res. 2000; 86: 1146-1152Google Scholar, 27Kron S.J. Toyoshima Y.Y. Uyeda T.Q. Spudich J.A. Methods Enzymol. 1991; 196: 399-416Google Scholar), wild type troponin increased sliding velocity to 125 ± 2.6% that of actin-tropomyosin filaments. However, filaments reconstituted with troponin containing ΔLys-210 troponin T gave a significantly reduced sliding speed (111 ± 2.7% of actin-tropomyosin), and this difference was highly reproducible (p <0.0001, 11 separate experiments, Fig. 1 B). Troponin containing ΔLys-210 mutant troponin T also induced a small but significant decrease (p <0.05) in the fraction of filaments motile under activating conditions compared with wild type troponin (Figs. 1 C and 2 B). Gel electrophoresis of the reconstituted thin filaments showed that both wild type and mutant thin filaments contained a full complement of bound troponin subunits and tropomyosin (Fig. 2 C). Under relaxing conditions (pCa9), ATPase activation and filament motility of thin filaments containing wild type and ΔLys-210 mutant troponin T were indistinguishable. ATPase activity was inhibited by ∼90% (Fig. 1 A), and in vitro motility was switched off by troponin such that <10% of the filaments were moving, and the speed of the remaining moving filaments was about 40% slower than actin-tropomyosin filaments, in agreement with previous measurements (24Redwood C.S. Lohmann K. Bing W. Esposito G.M. Elliott K. Abdulrazzak H. Knott A. Purcell I. Marston S.B. Watkins H. Circ. Res. 2000; 86: 1146-1152Google Scholar, 29Burton D. Abdulrazzak H. Knott A. Elliott K. Redwood C. Watkins H. Marston S. Ashley C. Biochem. J. 2002; 362: 443-451Google Scholar) using this system (Fig. 1, B andC). The maximum switch-off of motility required 10–15 nm troponin with both wild type and ΔLys-210 mutant troponin T. Comparing data from paired preparations of thin filaments, we found no significant difference in the fraction of filaments moving at saturating troponin concentrations (Fig. 1 C) nor at partially saturating concentrations (2.5–5 nm troponin). These results demonstrate that neither the EC50 of inhibition caused by troponin containing ΔLys-210 mutant troponin T, nor the maximum inhibition of motility, was different from wild type. Thin filaments reconstituted with troponin containing the HCM troponin T mutation R92Q reproducibly gave enhanced activation of ATPase activity (224 ± 5% of actin-tropomyosin activity compared with 207 ± 3% using wild type) and thin filament sliding velocity (Fig. 1, A and B). In addition, atpCa9 thin filaments containing R92Q troponin T reproducibly gave less inhibition of ATPase (79% compared with 90% using wild type troponin) and less inhibition of filament motility than wild type (p = <0.05; see Fig. 1, A andC). We determined the Ca2+ concentration dependence of thin filament activation of S-1 ATPase activity and motility using concentrations of troponin that gave maximal inhibition of filament activity at pCa9 (1 μm for ATPase, 15 nm for motility assay; Fig. 3and Table I). A consistent pattern of results was obtained with both techniques: the Ca2+-dependent curves for ATPase activation, fraction of filaments motile, and filament velocity of thin filaments containing ΔLys-210 mutant troponin T were much less steep (i.e. less cooperative) than those obtained using wild type troponin, and 50% activation was shifted to lower Ca2+concentrations. The data were fitted to the Hill equation:rate = a + b/(1 + 10nH(pCa-pCa50)). The ΔLys-210 mutation increased calculated pCa50by 0.19 ± 0.03, 0.28 ± 0.04, and 0.31 ± 0.03pCa units for ATPase, fraction of filaments motile, and sliding speed, respectively. The apparent Hill coefficients,n H, derived from fits of the mutant troponin data were close to unity (n H = 1.24 ± 0.06, 0.88 ± 0.19, and 0.90 ± .12, respectively) and the ATPase data were well fitted by a simple saturation equation (rate =V max[Ca2+]/(EC50+[Ca2+]) with EC50 = 0.25 ± 0.03 μm), indicating that these thin filaments were not cooperatively activated by Ca2+. In contrast, the R92Q troponin T mutation had a different effect; the Ca2+ sensitivity of ATPase regulation was increased with no change in cooperativity (Table I).Table IpCa50 values and Hill coefficients (nH) for the Ca2+ activation of thin filaments containing wild type and mutant troponinATPasepCa50n HSliding velocitypCa50n HMotile fraction pCa50n HWild type troponin T6.48 ± 0.02 (n= 8)2.98 ± 0.18 (n = 8)6.59 ± 0.05 (n = 3)1.65 ± 0.13 (n = 3)6.75 ± 0.13 (n = 3)1.85 ± 0.46 (n = 3)ΔLys-210 troponin T6.67 ± 0.01 (n = 8)1.24 ± 0.06 (n = 8)6.90 ± 0.08 (n= 3)0.90 ± 0.12 (n = 3)7.03 ± 0.15 (n = 3)0.88 ± 0.19 (n = 3)R92Q troponin T6.72 ± 0.03 (n = 8)2.97 ± 0.38 (n= 8)NDaNot determined.NDaNot determined.NDaNot determined.NDaNot determined.ATPase activities were measured at 37 °C, pH 7.0, and motility was measured at 28 °C, pH 7.4. Data from individual experiments were fitted to the Hill equation, and the means ± S.E. of the derived parameters, pCa50 and n H, fromn separate experiments are given in the table.a Not determined. Open table in a new tab ATPase activities were measured at 37 °C, pH 7.0, and motility was measured at 28 °C, pH 7.4. Data from individual experiments were fitted to the Hill equation, and the means ± S.E. of the derived parameters, pCa50 and n H, fromn separate experiments are given in the table. The disease caused by both the DCM and HCM troponin T mutations is autosomal dominant, and it is likely that the cardiac thin filaments of affected individuals contain similar proportions of wild type and mutant troponin. Our previous work has shown that the effect on thin filament function of mixtures of wild type and mutant troponin is not directly predictable from the functional properties of the mutant troponin alone (24Redwood C.S. Lohmann K. Bing W. Esposito G.M. Elliott K. Abdulrazzak H. Knott A. Purcell I. Marston S.B. Watkins H. Circ. Res. 2000; 86: 1146-1152Google Scholar, 25Elliott K. Watkins H. Redwood C.S. J. Biol. Chem. 2000; 275: 22069-22074Google Scholar). Thin filaments were reconstituted using stoichiometric amounts (7:1:1, respectively) of actin, tropomyosin, and total troponin (either wild type, mutant, or 50:50 wild type mutant). Filaments containing an eqimolar mix of wild type and mutant gave activation relative to actin-tropomyosin at pCa4.5 that was similar to the level using 100% mutant complex and significantly less (p <0.001) than that obtained with wild type troponin (Fig.4 A). Similarly, in thein vitro motility assay the speed of sliding of a 50:50 mixture was close to that of 100% mutant (2.31 ± 0.04, 2.34 ± 0.06, 2.53 ± 0.01 μm/sec for 100% mutant, 50:50 mixture, and 100% wild type, respectively). In contrast, thin filaments reconstituted with an equimolar mix of wild type troponin T and R92Q mutant troponin T gave similar levels of activation and inhibition as 100% wild type (Fig. 4 B). Interestingly, whereas 100% ΔLys-210 troponin T mutant gave an increase in Ca2+ sensitivity of ATPase activation of +0.23pCa units, the ΔLys-210/wild type mixture resulted in a significant decrease in Ca2+ sensitivity compared with wild type; ΔpCa50 = −0.27 (p <0.001;n = 4) (Fig. 4 A). The apparent Hill coefficient for the 50:50 mixture was intermediate between 100% wild type and 100% mutant and was significantly different from that obtained with wild type troponin alone (p <0.001). The same pattern of results was observed using in vitro motility assay: in thin filaments with a 50:50 mixture of wild type and ΔLys-210 troponin, the Ca2+ sensitivity of the fraction motile parameter was less than wild type by 0.07 pCa units (data not shown). Filaments containing an equimolar mix of wild type and R92Q troponin T produced an increase in the Ca2+ sensitivity of regulation of ATPase activation that was intermediate between pure wild type and pure mutant filaments with no change in cooperativity (ΔpCa50 = +0.10 for 50:50 wild type/R92Q compared with ΔpCa50 = +0.18 for R92Q) (Fig.4 B). The deletion of lysine 210 in cardiac troponin T has been reported to be a cause of inherited dilated cardiomyopathy (6Kamisago M. Sharma S.D. DePalma S.R. Solomon S. Sharma P. McDonough B. Smoot L. Mullen M.P. Woolf P.K. Wigle E.D. Seidman J.G. Seidman C.E. Jarcho J. Shapiro L.R. N. Engl. J. Med. 2000; 343: 1688-1696Google Scholar). When human cardiac troponin T with this mutation was incorporated into reconstituted thin filaments, we found a pattern of functional changesin vitro that was distinctly different from the changes previously observed with hypertrophic cardiomyopathy mutations. The Ca2+-activated rate of actomyosin ATP hydrolysis and the thin filament sliding speed were reduced compared with wild type troponin, the Ca2+ activation curve became non-cooperative, and pCa50 was increased. It is particularly noteworthy that a 50:50 mixture of wild type and mutant troponin T, which is likely to reflect the situation in vivo, still reduced the maximally activated actomyosin ATPase and filament sliding speed to the same level as 100% mutant troponin T but gave Ca2+ sensitivity significantly lower than wild type. The deleted amino acid forms one of a stretch of four lysine residues in human cardiac troponin T (amino acids 207–210). These amino acids lie within the C-terminal chymotryptic T2 fragment known to bind tropomyosin, troponin C, and troponin I (17Perry S.V. J. Muscle Res. Cell Motil. 1998; 19: 575-602Google Scholar). Studies of peptides and deletions within this region have indicated that troponin I binds to heptad repeat sequences C-terminal to these four lysines (approximately residues 229–268) (30Stefancsik R. Jha P.K. Sarkar S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 957-962Google Scholar), whereas both troponin C and tropomyosin may interact close to or directly with these residues (31Filatov V.L. Katrukha A.G. Bulargina T.V. Gusev N.B. Biochemistry (Mosc.). 1999; 64: 969-985Google Scholar). The recently reported (32Takeda S. Yamashita A. Maeda K. Maeda Y. Biophys. J. 2002; 82: 170aGoogle Scholar) crystal structure of the T2 fragment in complex with troponins I and C shows that residue 210 forms part of a short α-helix N-terminal to the separate helix involved in a coiled coil interaction with troponin I that has no direct association with any other troponin subunit. The change in maximum sliding speed and ATPase is compatible with experiments that have shown that one function of troponin T is to determine the cross-bridge turnover rate (12Sweeney H.L. Feng H.S. Yang Z. Watkins H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14406-14410Google Scholar, 26Bing W. Fraser I.D.C. Marston S.B. Biochem. J. 1997; 327: 335-340Google Scholar, 33Potter J.D. Sheng Z. Pan B.S. Zhao J. J. Biol. Chem. 1995; 270: 2557-2562Google Scholar); the remarkable uncoupling of cooperative activation could be related to the role of troponin T in determining the size of the thin filament cooperative unit through its interaction with tropomyosin (34Geeves M.A. Lehrer S.S. Biophys. J. 1994; 67: 273-282Google Scholar, 35Schaertl S. Lehrer S.S. Geeves M.A. Biochemistry. 1995; 34: 15890-15894Google Scholar). The recent report of a DCM mutation within the N-terminal T1 domain of troponin T, which only binds to tropomyosin (36Li D. Czernuszewicz G.Z. Gonzalez O. Tapscott T. Karibe A. Durand J.B. Brugada R. Hill R. Gregoritch J.M. Anderson J.L. Quinones M. Bachinski L.L. Roberts R. Circulation. 2001; 104: 2188-2193Google Scholar), and two mutations in tropomyosin itself (8Olson T.M. Kishimoto N.Y. Whitby F.G. Michels V.V. J. Mol. Cell Cardiol. 2001; 33: 723-732Google Scholar) also suggest that alterations in tropomyosin-troponin T interactions may be responsible for the appearance of the DCM phenotype (reviewed in Ref. 16Marston S.B. Hodgkinson J.L. J. Muscle Res. Cell Motil. 2001; 22: 1-4Google Scholar). The decreased Ca2+ sensitivity observed with 50:50 wild type/ΔLys-210 mixtures was surprising given the significant increase in Ca2+ sensitivity obtained in experiments using 100% DCM mutant troponin T. This emphasizes our previous findings that the regulatory properties of a 50:50 wild type/mutant troponin mixture are often quite different from both wild type and mutant troponin in a way that could not be predicted from the functional properties of the mutant troponin alone (24Redwood C.S. Lohmann K. Bing W. Esposito G.M. Elliott K. Abdulrazzak H. Knott A. Purcell I. Marston S.B. Watkins H. Circ. Res. 2000; 86: 1146-1152Google Scholar, 25Elliott K. Watkins H. Redwood C.S. J. Biol. Chem. 2000; 275: 22069-22074Google Scholar, 29Burton D. Abdulrazzak H. Knott A. Elliott K. Redwood C. Watkins H. Marston S. Ashley C. Biochem. J. 2002; 362: 443-451Google Scholar). The laboratory of Morimoto has recently reported a similar decreased Ca2+ sensitivity in rabbit heart trabeculae in which endogenous troponin was displaced upon incubation with human troponin T (either wild type or mutant) and human complex reconstituted in situ by the addition of human troponins I and C (37Morimoto S., Lu, Q.W. Harada K. Takahashi-Yanaga F. Minakami R. Ohta M. Sasaguri T. Ohtsuki I. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 913-918Google Scholar). It appears that these treated trabeculae contained about 50% human mutant troponin T and 50% endogenous rabbit troponin T. Decreased Ca2+ sensitivity together with the decreased cross-bridge turnover rate and reduced cooperativity suggest that the expression and incorporation of this DCM troponin T mutantin vivo may result in myofilaments that are markedly less responsive to Ca2+, contract more slowly, and thus are unable to produce sufficient force during activation. Interestingly, the changes in contractility predicted to be caused by the DCM troponin T mutant closely resemble the alterations in myofilament regulation identified in end stage failing human hearts. Investigations using tissue from hearts affected by a number of aetiologies (including non-familial DCM) have shown a consistent pattern of reduced unloaded shortening speed and reduced myofibrillar ATPase activity (38Hajjar R.J. Grossman W. Gwathmey J.K. Basic. Res. Cardiol. 1992; 87 Suppl. 1: 143-159Google Scholar, 39Ruf T. Schulte-Baukloh H. Ludemann J. Posival H. Beyersdorf F. Just H. Holubarsch C. Cardiovasc. Res. 1998; 40: 580-590Google Scholar, 40de Tombe P.P. Cardiovasc. Res. 1998; 37: 367-380Google Scholar). Reports are less clear over possible changes in Ca2+ sensitivity that has been suggested to be increased or unaltered; however, recent work suggests that additional factors such as sarcomere length and phosphate concentration are involved in determining which way the Ca2+ sensitivity will change (41Wolff M.R. Buck S.H. Stoker S.W. Greaser M.L. Mentzer R.M. J. Clin. Invest. 1996; 98: 167-176Google Scholar, 42Hajjar R.J. Schwinger R.H. Schmidt U. Kim C.S. Lebeche D. Doye A.A. Gwathmey J.K. Circulation. 2000; 101: 1679-1685Google Scholar, 43van der Velden J. Klein L.J. Zaremba R. Boontje N.M. Huybregts M.A. Stooker W. Eijsman L. de Jong J.W. Visser C.A. Visser F.C. Stienen G.J. Circulation. 2001; 104: 1140-1146Google Scholar). The reduced unloaded shortening speed correlates with the reduced ATPase and sliding speed caused by the ΔLys-210 mutation in troponin T. The effects of the ΔLys-210 mutation also correlate with the altered properties of troponin extracted from failing hearts. Failing heart troponin was found to confer a slower maximum sliding speed and a higher Ca2+ sensitivity with reduced cooperativity (22Purcell I.F. Bing W. Marston S.B. Cardiovasc. Res. 1999; 43: 884-891Google Scholar, 23Knott A. Purcell I.F. Marston S.B. J. Mol. Cell. Cardiol. 2002; 34: 469-482Google Scholar). Thus it is possible that a reduced maximum rate of cross-bridge turnover is a common causative feature of end stage heart failure, because a defect of this kind could not be fully compensated for by increasing Ca2+ sensitivity or modifying Ca2+ handling (44Perez N.G. Hashimoto K. McCune S. Altschuld R.A. Marban E. Circulation. 1999; 99: 1077-1083Google Scholar). It remains unclear whether these contractility changes in end stage failing hearts mediate the progress of heart failure or whether they are compensatory epiphenomena. However, the data presented here that show a troponin T mutant that is responsible for inherited DCM causing similar changes in contractility supports the notion that alterations to troponin may contribute directly to the progression of acquired forms of DCM. The functional alterations caused by the ΔLys-210 troponin T mutation are in many respects opposite from the data presented here for the R92Q HCM mutant and from our analyses of other HCM troponin T mutants (Ref. 24Redwood C.S. Lohmann K. Bing W. Esposito G.M. Elliott K. Abdulrazzak H. Knott A. Purcell I. Marston S.B. Watkins H. Circ. Res. 2000; 86: 1146-1152Google Scholar). 3P. Robinson, H. Watkins, and C. Redwood, unpublished data. Furthermore, this combination of properties is distinct from any other reported in vitro biochemical study on HCM troponin T mutants (21Szczesna D. Zhang R. Zhao J. Jones M. Guzman G. Potter J.D. J. Biol. Chem. 2000; 275: 624-630Google Scholar, 45Tobacman L.S. Lin D. Butters C. Landis C. Back N. Pavlov D. Homsher E. J. Biol. Chem. 1999; 274: 28363-28370Google Scholar, 46Knollmann B.C. Potter J.D. Trends Cardiovasc. Med. 2001; 11: 206-212Google Scholar), suggesting that the reduced maximal activation, depressed cooperativity, and, at an equimolar ratio with wild type troponin, diminished Ca2+ sensitivity may provide a specific stimulus for the production of the dilated, rather than hypertrophic, phenotype. We thank Chris Ashley, Hend Farza, and Kate Smith for discussions on these data.