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Ca2؉-desensitizing effect of a deletion mutation ⌬K210 in cardiac T that causes familial dilated

S. Morimoto*†‡, Q.-W. Lu*‡, K. Harada*‡§, F. Takahashi-Yanaga*‡, R. Minakami¶, M. Ohta*ʈ, T. Sasaguri*, and I. Ohtsuki*

*Laboratory of Clinical Pharmacology, Department of Pharmacology, Graduate School of Medicine, and ¶School of Health Sciences, Kyushu University, Fukuoka 812-8582, Japan

Communicated by Setsuro Ebashi, National Institute for Physiological Sciences, Okazaki, Japan, November 26, 2001 (received for review August 27, 2001) A deletion mutation ⌬K210 in cardiac (cTnT) was reported Ca2ϩ-sensitizing effects of the HCM-linked mutations recently found to cause familial (DCM). To in cTnT and cTnI. Tobacman et al. (17) reported that ⌬E160 ϩ explore the effect of this mutation on contraction cTnT mutant increased the Ca2 sensitivity of acto-S1 MgAT- ,under physiological conditions, we determined the Ca2؉-activated Pase activity. Szczesna et al. (18) reconstituted I79N, R92Q force generation in permeabilized rabbit cardiac muscle fibers into F110I, and R278C cTnT mutants into skinned cardiac muscle ϩ which the mutant and wild-type cTnTs were incorporated by using fibers and reported that these mutations increased Ca2 sensi- our TnT exchange technique. The free Ca2؉ concentrations re- tivity. Redwood et al. (19) also reported that one of the two 2ϩ quired for the force generation were higher in the mutant cTnT- truncated cTnT mutants had a Ca -sensitizing effect on acto-S1 exchanged fibers than in the wild-type cTnT-exchanged ones, with MgATPase activity. Recently, Miller et al. (20) and Chandra et no statistically significant differences in maximal force-generating al. (21) reported that transgenic mice expressing I79N and R92Q 2ϩ capability and cooperativity. Exchanging the mutant cTnT into cTnT mutants did exhibit increased Ca sensitivity in skinned 2؉ muscle fibers. Elliott et al. (22) reported that R145G and R162W isolated cardiac myofibrils also increased the free Ca concentra- 2ϩ tions required for the activation of ATPase. In contrast, a deletion mutations in cTnI increased the Ca sensitivity of - -activated S-1 ATPase activity, and James et mutation ⌬E160 in cTnT that causes familial hypertrophic cardio- ؉ al. (23) reported that transgenic mice expressing R145G (R146G myopathy (HCM) decreased the free Ca2 concentrations required ϩ in the mouse sequence) cTnI mutant exhibit increased Ca2 for force generation, just as in the case of the other HCM-causing sensitivity in skinned muscle fibers. These studies strongly mutations in cTnT. The results indicate that cTnT mutations found suggest that Ca2ϩ sensitization of force generation in in the two distinct forms of cardiomyopathy (i.e., HCM and DCM) is a primary mechanism for the pathogenesis of HCM with the -change the Ca2؉ sensitivity of cardiac in oppo ؉ mutations in Tn subunits (24, 25). Recently, however, a novel site directions. The present study strongly suggests that Ca2 mutation (⌬K210) in the cTnT was found to cause a quite desensitization of force generation in sarcomere is a primary different form of cardiomyopathy, dilated cardiomyopathy mechanism for the pathogenesis of DCM associated with the (DCM), which is characterized by cardiac dilation and reduced MEDICAL SCIENCES deletion mutation ⌬K210 in cTnT. systolic function leading to failure with high mortality (26). In the present study, an attempt was made to directly exchange ⌬ ontraction of the vertebrate-striated muscles (i.e., skeletal the recombinant human K210 cTnT into membrane- Cand cardiac muscles) is regulated by Ca2ϩ through its binding permeabilized (skinned) rabbit cardiac muscle fibers. This tech- to a specific regulatory complex, troponin (Tn), which is nique overcomes the potentially significant complications distributed at regular intervals along the entire thin filament (1, caused by compensatory mechanisms expected to occur when 2). Tn is a complex of three different , troponin T (TnT; transgenesis is used in a whole animal. The study revealed that the functional consequence of the mutation ⌬K210 in cTnT is a tropomyosin-binding component), (TnI; inhibitory 2ϩ ϩ decrease in the Ca sensitivity of cardiac muscle contraction. component), and (TnC; Ca2 -binding component). 2ϩ 2ϩ The results strongly suggest that the primary mechanism for the On Ca binding to TnC, a Ca -induced interaction of TnC pathogenesis of DCM associated with the mutation ⌬K210 is a with TnI relieves the inhibitory action of TnI exerted on the thin deficiency of force generation by sarcomere in cardiac muscle, in filament and enables the myosin head to cyclically interact with 2ϩ contrast to the enhancement of force generation in HCM actin in the thin filament and generate force. The Ca sensitivity associated with the other mutations in cTnT. of muscle contraction is determined by the Ca2ϩ-binding affinity of TnC, which is dynamically altered through interaction with Materials and Methods TnI and TnT in the lattice (3–8). Mutagenesis of Recombinant Human cTnT. The cloning and mu- Mutations in for cardiac troponin T (cTnT) and cardiac tagenesis of human cTnT cDNA were carried out as described troponin I (cTnI) have been found to cause familial hypertrophic cardiomyopathy (HCM), an autosomal dominant heart disease characterized by asymmetrical with a Abbreviations: Tn, troponin; cTnT, cardiac Tn T; cTnI, cardiac Tn I; HCM, hypertrophic ϩ high incidence of sudden death in young adults (9). We have cardiomyopathy; DCM, dilated cardiomyopathy; pCa, Ϫlog[Ca2 ]. already examined the effects of eight HCM-linked cTnT muta- †To whom reprint requests should be addressed. E-mail: [email protected] tions (I79N, R92Q, ⌬E160, E244D, R278C, and two truncated u.ac.jp. 3 ‡S.M., Q.-W.L., K.H., and F.T.-Y. contributed equally to this work. mutants produced by a splice donor site mutation Int15G1 A) and six HCM-linked cTnI mutations (R145G, R145Q, R162W, §Present address: Department of Molecular and Cellular Pharmacology, University of Miami ⌬K183, G203S, and K206Q) on the contractile functions of School of Medicine, Miami, FL 33136. ʈ cardiac muscle by using a technique for exchanging the exoge- Postdoctoral fellow on leave of absence from Laboratory for Structural Biochemistry, The Institute of Physical and Chemical Research, Harima Institute at Spring-8, Hyogo 679-5148, nous Tn complex into skinned muscle fibers and isolated myo- Japan. 2ϩ fibrils. We found that Ca sensitization in cardiac muscle The publication costs of this article were defrayed in part by page charge payment. This contractility is a common effect caused by HCM-linked muta- article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. tions in cTnT and cTnI (10–16). Several groups also have §1734 solely to indicate this fact.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.022628899 PNAS ͉ January 22, 2002 ͉ vol. 99 ͉ no. 2 ͉ 913–918 Downloaded by guest on September 24, 2021 structed into the pET-3d vector. The complete nucleotide se- quences of the mutant cTnT cDNAs were confirmed by DNA sequencing.

Preparation of Skinned Fibers and Force Measurements. Rabbit cardiac skinned muscle fibers were prepared from the left ventricular trabeculae of young male albino rabbits (Ϸ3 mo old) as described (10). In brief, small bundles (0.5–1 mm wide and 5–7 mm long) of trabeculae tied to glass capillary tubes were skinned with relaxing solution containing 0.5% Brij-58 for 30 min at 25°C and were stored up to 3 wk at Ϫ20°C in relaxing solution containing 50% glycerol. A small fiber (Ϸ120 ␮m in diameter) dissected from the stock-skinned trabecula was mounted in a thermostatically controlled chamber with a capacity of 0.2 ml. Fiber length between hooks was Ϸ1 mm, and the resting sarcomere length was set to 2.3 ␮m by using laser diffraction. Fig. 1. SDS͞PAGE of recombinant human cTnT and native rabbit cardiac Tn The force generated by skinned muscle fibers was measured at three components. Lane 1, rabbit skinned cardiac muscle; lane 2, recombinant 25°C with a strain gauge, UL-2GR (Minebea, Japan). The human wild-type cTnT; lane 3, recombinant human ⌬K210 cTnT; lane 4, native ͞ rabbit cTnT; lane 5, native rabbit cTnI, and lane 6, native rabbit cTnC. The gel relaxing solution consisted of (in mM) 50 Mops KOH (pH 7.0), was stained with Coomassie brilliant blue R-250. 100 KCl, 6 MgCl2, 5 ATP, 4 EGTA, 0.5 DTT, and 10 creatine phosphate, as well as 35 units͞ml . Activating solutions with desired free Ca2ϩ concentrations were prepared previously (14). To generate the ⌬K210 and ⌬E160 cDNAs, by adding appropriate amounts of CaCl2, calculated as described mutageneses were first carried out by PCR according to the (28), to the relaxing solution. GeneSOEing method described by Horton (27) by using the following oligonucleotide primers: 5Ј-GGT GGT GGA AGC ATPase Activity Measurement. Porcine cardiac were iso- GTA CGA AGA GG-3Ј (18F, SplI site is underlined), 5Ј-CCT lated from left ventricular muscle, and their ATPase activity was CTC AGC CAG AAT CTT CTT CTT TTC CCG (645R), measured in a reaction mixture (150 ␮l) that consisted of 90 mM ͞ 2ϩ 5Ј-AAG AAG ATT CTG GCT GAG AGG AGG AAG GTG KCl, 5 mM MgCl2, 20 mM Mops KOH (pH 7.0), 1 mM Ca - (622F), and 5Ј-GCT GCA GGA TCC TAT TTC CAG CGC EGTA, 4 mM ATP, and 45 ␮g of myofibrils, as described (13). CCG G (878 R, BamHI site is underlined) for ⌬K210; 5Ј-GGT GGT GGA AGC GTA CGA AGA GG-3Ј (18F, SplI site is Purification of Proteins. Expression and purification of the recom- underlined), 5Ј-CCT GTT CTC CTC CTC TCG TCG AGC CCT binant human cTnTs were performed as described previously CTC (495R), 5Ј-CGA CGA GAG GAG GAG AAC AGG AGG (10). The wild-type and mutant cTnTs were eluted at 0.3–0.4 M AAG GCT (472F), and 5Ј-GCT GCA GGA TCC TAT TTC NaCl on a fast-performance liquid chromatography (FPLC) CAG CGC CCG G (878 R, BamHI site is underlined) for ⌬E160. ion-exchange column, RESOURCE Q (6 ml) (Amersham Phar- The obtained PCR products digested by SplI and BamHI were macia Biotech), with a linear gradient of 0–0.5 M NaCl in the ligated to a NcoI linker made from oligonucleotides 5Ј-CAT presence of 20 mM Tris⅐HCl (pH 8.0)͞6 M urea͞5 mM trans-1, GTC TGA CAT CGA AGA AGT GGT GGA AGA and 5Ј-GTA 2-cyclohexanediamine-N,N,NЈ,NЈ-tetraacetic acid͞and 15 mM CTC TTC CAC CAC TTC TTC GAT GTC AGA and con- 2-mercaptoethanol. Rabbit native cTnT, cTnI, and cTnC were

Fig. 2. SDS͞PAGE of rabbit skinned cardiac muscle fibers before and after exchanging endogenous cTnT with recom- binant human cTnT. Lane 1, purified rabbit cardiac Tn com- plex; lane 2, a control muscle fiber; lane 3, a muscle fiber treated with human wild-type cTnT; lane 4, a muscle fiber treated with human ⌬K210 cTnT; lane 5, a muscle fiber treated with human wild-type cTnT and reconstituted with rabbit cTnI and cTnC, and lane 6, a muscle fiber treated with human ⌬K210 cTnT and reconstituted with rabbit cTnI and cTnC. The gel was stained with silver. wt, wild-type; tr., treatment; MHC, myosin heavy chain; Tm, tropomyosin; LC1, 1; and LC2, myosin light chain 2.

914 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.022628899 Morimoto et al. Downloaded by guest on September 24, 2021 MEDICAL SCIENCES

Fig. 3. Stoichiometry of Tn components in rabbit skinned cardiac muscle fibers after treatment (tr.) with recombinant human cTnT and after subsequent reconstitution with rabbit cTnI and cTnC (ϩ TnI⅐TnC). SDS͞PAGE gels shown in Fig. 2 were analyzed by an optical densitometric scan. The amounts of cTnI, cTnC, and cTnT were normalized to the amount of LC1 in each muscle fiber and are expressed as means Ϯ SEM of three independent experiments.

prepared from the left ventricular myocardium of young male Data Reduction and Error Analysis. To determine the pCa albino rabbits (Ϸ3 mo old) according to the method of Tsukui (Ϫlog[Ca2ϩ]) value at half-maximal force generation or ATPase and Ebashi (29) by using FPLC ion-exchange columns, RE- activation (pCa50) and the Hill coefficient (nH), the force and SOURCE Q and S (6 ml), and Mono Q and S HR 5͞5 ATPase activity were normalized to the maximum force in the (Amersham Pharmacia Biotech). The purified proteins used in same fiber and to the maximum ATPase activity in the same the present study are shown in Fig. 1. series of measurement, respectively, and the relative force or ATPase–pCa relationship in each fiber or in each series of TnT Exchange in Skinned Fibers. cTnT exchange in the skinned ATPase measurement was fitted to the following form of the Hill fibers and isolated myofibrils was performed by the method equation by means of the Marquardt nonlinear least-squares described previously (30–32). method (GRAPHPAD PRISM Ver. 3.00 for Windows, GraphPad, San Diego): SDS͞PAGE Analysis. SDS͞PAGE was carried out at 12% acryl- amide concentration according to the method of Laemmli (33). Relative force or ATPase activity, % ͑ Ϫ ͒⅐ The fiber samples were lysed in Laemmli’s sample buffer by ϭ 100͕͞1 ϩ 10 pCa pCa50 nH͖. heating for 4 min at 95°C after freezing (Ϫ80°C) and thawing

several times. The gel was stained with silver by using a staining The mean values of pCa50 and nH and their SEM were then (Amersham Pharmacia Biotech) or with Coomassie brilliant calculated. The value r2, a measure of goodness of fit, was in the blue R-250. An optical densitometric scan was performed by range of 0.98–1.0 in all determinations. using PHORETIX gel analysis software (1-D Gel Analysis, Phoretix International, Newcastle upon Tyne, England) cali- Results brated by a photographic step tablet (21 steps; density range Fig. 1 shows SDS͞PAGE of the bacterially expressed and 0.05–3.03, Eastman Kodak). The linearity of the densitometric purified human wild-type and DCM-causing deletion mutant reading was confirmed by changing the amount of sample ⌬K210 cTnTs as well as the purified native rabbit cardiac Tn applied on the gel in steps. three subunits. Fig. 2 shows an SDS͞PAGE analysis of the rabbit

Morimoto et al. PNAS ͉ January 22, 2002 ͉ vol. 99 ͉ no. 2 ͉ 915 Downloaded by guest on September 24, 2021 Fig. 4. Effects of mutations in cTnT on force generation of cardiac muscle. (A) Force–pCa relationships in the rabbit skinned cardiac muscle fibers into which human wild-type, ⌬K210, or ⌬E160 cTnT was incorporated. (B) Effects of mutations on maximum force-generating capability. Data are expressed as percentage of the mean maximum force generated by fibers with wild-type cTnT. Maximum force levels after cTnT exchange were 72.9 Ϯ 3.3, 68.1 Ϯ 4.9, and 67.7 Ϯ 1.7% of the maximum force determined before exchange in the fibers with wild-type, ⌬K210, and ⌬E160 cTnTs, respectively. (C and D) Effects of mutations on Ca2ϩ sensitivity (pCa50) and steepness (nH) of the force–pCa relationships, respectively. The data represent the means Ϯ SEM of measurements on five, six, and five fibers for wild-type, ⌬K210, and ⌬E160 cTnTs, respectively. Statistical significance was determined by ANOVA followed by the post-hoc Dunnett’s multiple comparison test.

skinned cardiac muscle fibers before and after exchanging native the Ca2ϩ-sensitizing effect (10–12, 14, 15, 34). In contrast, the cTnT with recombinant human wild-type or ⌬K210 cTnT. force–pCa relationship in the ⌬K210 mutant cTnT-exchanged ϩ Treatment of skinned fibers with an excess amount of recom- fibers was shifted to higher Ca2 concentrations compared to that in the wild-type cTnT-exchanged fibers, indicating that the binant human cTnT resulted in a decrease in the amount of ϩ endogenous cTnI and cTnC (lanes 3 and 4) because of displace- deletion mutation ⌬K210 decreases the Ca2 sensitivity of force ment of the Tn complex with recombinant human cTnT, as generation in cardiac muscle. The maximum force-generating demonstrated in our previous studies (10, 14, 30–32). Subse- capabilities of these three fiber groups were not statistically quent incubation with rabbit cTnI and cTnC accomplished the significantly different (Fig. 4B). Curve fittings of the data in Fig. 4A to the Hill equation revealed that the pCa value at half- exchange of recombinant human cTnT into rabbit cardiac muscle 2ϩ maximal force generation (pCa50, an index of Ca sensitivity) fibers (lanes 5 and 6). Densitometric scans of the gels demon- ⌬ ⌬ strated that there was no difference in the extent of incorpora- was statistically significantly lower in K210 and higher in E160 mutation than in wild type, whereas the Hill coefficient values tion between wild-type and ⌬K210 cTnTs determined from the (n , an indicator of cooperativity or steepness of the curve) were decrease of cTnI and cTnC after cTnT treatment (P Ͼ 0.85, t H ⌬ not significantly different (Fig. 4 C and D). These results indicate test), and that exchange of wild-type and K210 cTnTs into that the deletion mutation ⌬K210 in cTnT has a Ca2ϩ- fibers did not change the stoichiometry of the three Tn compo- desensitizing effect on force generation in cardiac muscle with- nents in muscle fibers (Fig. 3). out changing the maximum force-generating capability and Fig. 4 compares the force–pCa relationship in the fibers ⌬ cooperativity. exchanged with the deletion mutant K210 with that in the fibers Next, we determined the ATPase–pCa relationship in the exchanged with wild-type cTnT. We also determined the force– ⌬K210 mutant cTnT-exchanged cardiac myofibrils (Fig. 5). The ⌬ ϩ pCa relationship in the E160 mutant cTnT-exchanged fibers as mutation ⌬K210 also decreased the Ca2 sensitivity of myofi- ⌬ a control. The mutation E160 is the only deletion mutation in brillar ATPase activity, as demonstrated by a rightward shift of cTnT associated with HCM, and we have demonstrated that this the ATPase–pCa relationship with a statistically significant 2ϩ mutation increases the Ca sensitivity of cardiac myofibrillar decrease in pCa50. The result clearly indicates that the deletion ATPase activity (13). As shown in Fig. 4A, the mutation ⌬E160 mutation ⌬K210 has a depressant effect on the Ca2ϩ regulatory ϩ shifted the force–pCa relationship to lower Ca2 concentrations, mechanism of cardiac muscle contraction at the level of acto- as is the case of the other HCM-linked cTnT mutations showing myosin interaction.

916 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.022628899 Morimoto et al. Downloaded by guest on September 24, 2021 evidence for this hypothesis has been provided experimentally, and no studies have been made on functional consequences of the DCM-causing mutations in these cytoskeletal and sarco- meric proteins. In the present study, we used a technique for exchanging Tn components in sarcomere, which made it possible to explore the functional change of mutated cTnT in muscle fiber with its contractile apparatus being kept intact. The deletion mutation ⌬K210 decreased the Ca2ϩ sensitivity of force generation with- out affecting the stoichiometry of three Tn components in sarcomere. This indicates that this mutation alters a relatively localized interaction within the Tn complex involving the Ca2ϩ- sensitive disinhibition of the TnI action by TnC. The lysine residue 210 in cTnT is in a domain that is involved in the Ca2ϩ-sensitive strong TnC binding and in the very weak tropo- myosin and TnI binding (46). Thus, the deletion mutation ⌬K210 might impair the Ca2ϩ-induced interaction of TnC with this region on cTnT. However, more precise structural information on the Tn complex from x-ray crystallography or NMR is required to elucidate the exact molecular mechanism by which the mutation causes the depression of Ca2ϩ sensitivity of force generation. The present study provides the first evidence, to our knowl- edge, that the decrease in Ca2ϩ sensitivity of force generation in sarcomere might be a primary mechanism for the pathogenesis of DCM in patients with the deletion mutation ⌬K210 in cTnT. This mutation does not affect the maximum force-generating capability of sarcomere. However, even a small decrease in Ca2ϩ sensitivity is expected to cause a significant reduction in the force generation of the cardiac muscle in the heart, because the intact cardiac muscle is known to never be activated beyond the half-maximal level (47). The reduction in force generation by sarcomere with the deletion mutation ⌬K210 in cTnT and the Fig. 5. Effects of ⌬K210 mutation in cTnT on ATPase activity of myofibrils. (A) deficits of force transmission in cytoskeletal proteins might go ATPase–pCa relationships in the rabbit isolated cardiac myofibrils into which through the same process of pathogenesis, leading to ventricular human wild-type or ⌬K210 cTnT was incorporated. (B) Effects of ⌬K210 dilation as a compensatory mechanism for the decrease in

2ϩ MEDICAL SCIENCES mutation on Ca sensitivity (pCa50) of ATPase–pCa relationships. The data Ϯ volume because of the reduction in contractile force of the heart. represent the means SEM of five independent experiments. Statistical 2ϩ significance was determined by the t test. The Hill coefficients of the ATPase– The decrease in Ca sensitivity of force generation also is pCa relationships are 1.76 Ϯ 0.13 and 1.39 Ϯ 0.16 for wild-type and ⌬210 cTnTs, expected to facilitate the relaxation of the cardiac muscle at respectively, which are not significantly different (t test). diastole and might directly explain the cardiac dilation itself to some extent. In contrast, we have previously shown that the mutations in cTnT associated with HCM have Ca2ϩ-sensitizing Discussion effects on force generation in cardiac muscle (10–15). The 2ϩ DCM represents a heterogeneous group of inherited and ac- increase in Ca sensitivity of force generation is expected to quired disorders characterized by cardiac dilation and systolic impair the relaxation of cardiac muscle and thus cause a diastolic dysfunction. Idiopathic DCM is a relatively common disorder dysfunction, which might be directly involved in the high inci- (Ϸ50% of DCM) that accounts for Ͼ10,000 deaths annually by dence of sudden death despite mild or no hypertrophy in HCM patients associated with cTnT mutations. Our studies indicate and sudden death in the United States (5-yr 2ϩ mortality ϭ 40Ϸ80%), being the primary indication for cardiac that the Ca sensitivity of force generation in cardiac muscle plays an extremely important role in cardiac function, and even transplantation (35–38). ϩ a sight change in the Ca2 sensitivity in either direction caused Recently, single gene defects that cause familial DCM (Ϸ25– by the mutations in the regulatory protein complex, troponin, 30% of idiopathic DCM) have been identified for cytoskeletal may have a fatal effect on the cardiac system. (, , tafazzin, and ␦-sarcogycan) and sarcomeric (actin, tropomyosin, myosin, and troponin T) proteins (26, This study was supported by Special Coordination Funds from the 39–45). Mutations in cytoskeletal proteins imply that deficits in Ministry of Education, Culture, Sports, Science and Technology, the transmission of force generated by sarcomere may be one Japanese Government. Q.-W.L. is an Uehara Memorial Foundation mechanism for the pathogenesis of DCM. However, no direct Research Fellow.

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