Ca2؉-desensitizing effect of a deletion mutation ⌬K210 in cardiac troponin T that causes familial dilated cardiomyopathy 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 troponin T (cTnT) was reported Ca2ϩ-sensitizing effects of the HCM-linked mutations recently found to cause familial dilated cardiomyopathy (DCM). To in cTnT and cTnI. Tobacman et al. (17) reported that ⌬E160 ϩ explore the effect of this mutation on cardiac muscle 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 actin- tropomyosin-activated myosin 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 sarcomere 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 muscle contraction 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 gene 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 heart 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 protein 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 proteins, 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), troponin I (TnI; inhibitory 2ϩ ϩ decrease in the Ca sensitivity of cardiac muscle contraction. component), and troponin C (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 myofilament lattice (3–8). Mutagenesis of Recombinant Human cTnT. The cloning and mu- Mutations in genes 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 ventricular hypertrophy 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 creatine kinase. 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 myofibrils 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).