Altered Regulation of Cardiac Muscle Contraction by Troponin T Mutations That Cause Familial Hypertrophic Cardiomyopathy*

To study the effect of troponin (Tn) T mutations that cause familial hypertrophic cardiomyopathy (FHC) on cardiac muscle contraction, wild-type, and the following recombinant human cardiac TnT mutants were cloned and expressed: I79N, R92Q, F110I, E163K, R278C, and intron 16(G1 → A) (In16). These TnT FHC mutants were reconstituted into skinned cardiac muscle preparations and characterized for their effect on maximal steady state force activation, inhibition, and the Ca2+ sensitivity of force development. Troponin complexes containing these mutants were tested for their ability to regulate actin-tropomyosin(Tm)-activated myosin-ATPase activity. TnT(R278C) and TnT(F110I) reconstituted preparations demonstrated dramatically increased Ca2+sensitivity of force development, while those with TnT(R92Q) and TnT(I79N) showed a moderate increase. The deletion mutant, TnT(In16), significantly decreased both the activation and the inhibition of force, and substantially decreased the activation and the inhibition of actin-Tm-activated myosin-ATPase activity. ATPase activation was also impaired by TnT(F110I), while its inhibition was reduced by TnT(R278C). The TnT(E163K) mutation had the smallest effect on the Ca2+sensitivity of force; however, it produced an elevated activation of the ATPase activity in reconstituted thin filaments. These observed changes in the Ca2+ regulation of force development caused by these mutations would likely cause altered contractility and contribute to the development of FHC.


From the Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Miami, Florida 33101
To study the effect of troponin (Tn) T mutations that cause familial hypertrophic cardiomyopathy (FHC) on cardiac muscle contraction, wild-type, and the following recombinant human cardiac TnT mutants were cloned and expressed: I79N, R92Q, F110I, E163K, R278C, and intron 16(G 1 3 A) (In16). These TnT FHC mutants were reconstituted into skinned cardiac muscle preparations and characterized for their effect on maximal steady state force activation, inhibition, and the Ca 2؉ sensitivity of force development. Troponin complexes containing these mutants were tested for their ability to regulate actin-tropomyosin(Tm)-activated myosin-ATPase activity. TnT(R278C) and TnT(F110I) reconstituted preparations demonstrated dramatically increased Ca 2؉ sensitivity of force development, while those with TnT(R92Q) and TnT(I79N) showed a moderate increase. The deletion mutant, TnT(In16), significantly decreased both the activation and the inhibition of force, and substantially decreased the activation and the inhibition of actin-Tm-activated myosin-ATPase activity. ATPase activation was also impaired by TnT(F110I), while its inhibition was reduced by TnT(R278C). The TnT(E163K) mutation had the smallest effect on the Ca 2؉ sensitivity of force; however, it produced an elevated activation of the ATPase activity in reconstituted thin filaments. These observed changes in the Ca 2؉ regulation of force development caused by these mutations would likely cause altered contractility and contribute to the development of FHC.
Vertebrate striated (skeletal and cardiac) muscle contraction is activated by the binding of Ca 2ϩ to the low affinity Ca 2ϩspecific (regulatory) sites of troponin (Tn) 1 C, the Ca 2ϩ -binding subunit of Tn, which together with TnI, TnT, and tropomyosin (Tm) form the regulatory system of the contractile apparatus (1)(2)(3)(4). Studies of the regulation of skeletal and cardiac muscle contraction have suggested that TnT not only anchors the TnI⅐TnC complex to the thin filaments through its interaction with TnI and Tm, but also confers the Ca 2ϩ sensitivity to the actomyosin-ATPase activity and/or force (5)(6)(7)(8).
In this study we have focused on the following FHC HCTnT mutations: TnT(I79N), TnT(R92Q), TnT(F110I), TnT(E163K), TnT(R278C), and TnT(In16). Several reports have appeared, which have examined the effects of these mutations on the biochemical and contractile properties of cardiac muscle (13,25,29,(31)(32)(33)(34). Four of the HCTnT mutants (I79N, R92Q, F110I, and R278C) studied in this paper have been reported in a recent paper from Yanaga et al. (34), who investigated their effect on myofibrillar ATPase activity in reconstituted myofibrils. In an earlier study, Morimoto et al. (32) reported on the effect of two of these mutations (I79N, R92Q) when incorporated into skinned cardiac trabeculae (rabbit). We have reconstituted the HCTnT mutants into actomyosin, and skinned cardiac (porcine) muscle fiber preparations and studied their effects on inhibition, activation, and the Ca 2ϩ sensitivity of ATPase and steady state force development. Our results are discussed in relation to those from the Ohtsuki lab (32,34) and others (31,33,35).
We found that TnT(R278C)-, TnT(F110I)-, TnT(I79N)-, and TnT(R92Q)-reconstituted skinned cardiac muscle preparations had significantly increased Ca 2ϩ sensitivity of force development, whereas those reconstituted with TnT(In16) had dramatically decreased maximal force and force inhibition. We also found that troponin complexes made from TnT(F110I) and TnT(In16) had substantially reduced activation of actin-Tmactivated myosin-ATPase activity while those containing TnT(R278C) and TnT(In16) demonstrated impaired inhibition of the ATPase. TnI and TnC-The  cDNAs encoding human cardiac TnT, TnI, and TnC were cloned by  reverse transcriptase-polymerase chain reaction using a template of  total RNA from human myocardium and oligonucleotide primers specific for the 5Ј and 3Ј regions of the respective coding sequences. Additionally, the six TnT FHC mutants: I79N, R92Q, F110I, E163K, R278C, and In16, a mutation in HCTnT, which arises from abnormal splicing of Intron 16(G 1 3 A), were made using a sequential overlapping polymerase chain reaction-based method (36). Standard methods were utilized for expression and purification of wild-type HCTnT and its FHC mutants as well as HCTnI and HCTnC (37-39). All clones were sequenced to verify the correct sequences prior to expression and purification of the respective proteins.

Mutation, Expression, and Purification of Wild-type HCTnT and HCTnT Mutants Expression and Purification of Human Cardiac
Actin-Tm-activated Myosin-ATPase Assays-Rabbit skeletal F-actin was prepared as described by Strzelecka et al. (40). Porcine cardiac myosin was purified according to Murakami et al. (41). Porcine cardiac Tm, TnC, and TnI were prepared from pig ventricles according to Potter (42). The complexes of wild-type HCTnT and its FHC mutants with porcine cardiac TnI and TnC were prepared according to the following protocol (42). All Tn subunits (T, I, and C) were first dialyzed against 6 M urea, 10 mM MOPS, pH 7.0, 0.25 mM CaCl 2 , 1 mM dithiothreitol, and 0.01% NaN 3 . After dialysis they were mixed at a molar ratio 1.25:1.25:1 (T:I:C) and incubated for 1 h on ice before being dialyzed against buffers with decreasing concentrations of urea. The dialysis buffer contained 1 M KCl, 10 mM MOPS, pH 7.0, 1 mM dithiothreitol, 0.01 NaN 3 and initially 6, then 3, followed by 0 M urea. Subsequently, the complexes were gradually dialyzed against buffers containing decreasing concentrations of KCl, starting from 1, then 0.7, 0.5, 0.3, and finally 0.1 M KCl. Excess of TnT and TnI that precipitated at the low ionic strength ϩ 0 M urea buffer was removed by centrifugation at 20,000 rpm for 30 min. The molar ratio of the reconstituted troponin complexes was verified by SDS 15%-PAGE (43) and was 1:1:1 for TnT:TnI:TnC.
The ATPase assays were performed with rabbit skeletal muscle F-actin (3.5 M) containing porcine cardiac Tm (1 M) and formed Tn complexes (1 M). The concentration of porcine cardiac myosin was 0.6 M. All ATPase assays were performed in the presence (0.5 mM CaCl 2 ) or absence (1 mM EGTA) of Ca 2ϩ . The ATPase reactions (in 10 mM MOPS, 50 mM KCl, 4 mM MgCl 2 , pH 7.0) were initiated with 2.5 mM ATP and after 20 min incubation, they were terminated with 5% trichloroacetic acid. Released inorganic phosphate was measured according to Fiske and SubbaRow (44).

Displacement of the Endogenous Tn Complex in Porcine Skinned Cardiac Muscle Preparations with HCWTnT and its Mutants: Steady-state Force and the Ca 2ϩ Sensitivity of Force Development
Porcine muscle fiber bundles were mounted on a force transducer and treated with the pCa 8 relaxing solution containing 1% Triton X-100 for ϳ1 h. The composition of the pCa 8 solution was 10 Ϫ8 M Ca 2ϩ , 1 mM Mg 2ϩ , 7 mM EGTA, 5 mM MgATP 2ϩ , 20 mM imidazole, pH 7.0, 20 mM creatinine phosphate, and 15 units/ml creatinine phosphokinase, I ϭ 150 mM. Subsequently, the fibers were transferred into the pCa 8 solution without Triton X-100 and then to the pCa 4 solution for the initial force determination. The composition of the pCa 4 solution was the same as that of the pCa 8 buffer, except the Ca 2ϩ concentration was 10 Ϫ4 M. To determine the Ca 2ϩ sensitivity of force development, the fibers were gradually exposed to the solutions of increasing Ca 2ϩ concentrations, from pCa 8 to pCa 4. To displace the endogenous Tn complex from the fibers they were incubated in a solution containing 250 mM KCl, 20 mM MOPS, pH 6.2, 5 mM MgCl 2 , 5 mM EGTA, 0.5 mM dithiothreitol, and Ϸ0.8 -1 mg/ml HCWTnT or its FHC mutants, for 1 h at room temperature. A fresh TnT protein was applied to the fibers for another 1-h incubation. This was to increase the efficiency of the endogenous Tn displacement from the fibers. Displaced fibers were then washed with the same solution without the protein (10 min at room temperature) and tested for Ca 2ϩ -unregulated force that developed due to the absence of the endogenous porcine cardiac TnI and TnC. The Ca 2ϩ regulation of steady-state force was restored with a preformed HCTnI⅐HCTnC complex. The reconstitution with the HCTnI⅐HCTnC complex (30 M) was performed in the pCa 8 solution for ϳ1.5 h at room temperature, or long enough for the force to reach a stable level. Control fibers were run in parallel and treated with the same solutions minus the proteins. The final Ca 2ϩ sensitivity of force development was determined after HCTnI⅐HCTnC reconstitution and the data were analyzed with the Hill equation: % relative force ϭ 100 ϫ [Ca 2ϩ ] nH /([Ca 2ϩ ] nH ϩ pCa 50 nH ), where pCa 50 determines the pCa of a solution in which 50% of a change is produced and n H is the Hill coefficient.

RESULTS
In this study we have characterized the following TnT mutations associated with familial hypertrophic cardiomyopathy: I79N, R92Q, F110I, E163K, R278C, and In16 mutation, which arises from abnormal splicing of intron 16 and results in a truncated TnT protein. These TnT mutants together with other Tn subunits (TnC and TnI) were reconstituted in actin-Tm containing thin filaments and tested for the Ca 2ϩ regulation of the actomyosin-ATPase activity. These mutants were also reconstituted in skinned cardiac muscle preparations and investigated for their ability to regulate force development, relaxation, and the Ca 2ϩ sensitivity of force development. Fig. 1 illustrates the amino acid sequence of the adult isoform (HCTnT3) of human cardiac TnT. The cDNA encoding HCTnT isoform lacks a cassette of 30 base pairs located in the 5Ј end of the molecule (45) and the resulting protein sequence is shorter by 10 amino acids compared with the embryonic isoform (HCTnT1). All identified FHC TnT mutations are boxed. Fig. 2 represents the exon organization of the full size HCTnT (isoform HCTnT1) (45,46). The FHC TnT mutations are also illustrated here, and correspond to the amino acid sequence of HCTnT3.
Tn Displacement of Skinned Cardiac Muscle Fibers with FHC TnT Mutants-The physiological significance of the FHC TnT mutations was examined utilizing porcine skinned fibers, in which steady-state force activation, inhibition, and the Ca 2ϩ sensitivity of force development were measured. The skinned cardiac muscle preparations were incubated with HCWTnT and its FHC mutants until a complete loss of the Ca 2ϩ dependence of force was observed. Fig. 4 illustrates an experimental protocol for TnT(E163K)-treated preparations. The same protocol was applied for HCWTnT and the other FHC TnT mutants. As shown in Fig. 4, incubation of the fibers with TnT(E163K) for 2 h at room temperature resulted in a complete loss of the Ca 2ϩ dependence of force following displacement of the Tn complex with TnT(E163K). The fibers became insensitive to Ca 2ϩ and could not relax due to the absence of the TnI⅐TnC complex. The level of Ca 2ϩ -unregulated force following the TnT treatment did not depend on the FHC TnT mutant utilized in the displacement procedure and was equal Ϸ60% of the force developed by the untreated skinned fiber preparations. Since the average rundown of the control fibers treated with the buffers minus proteins for the same amount of time as the experimental fiber was Ϸ12-18%, the true level of Ca 2ϩunregulated force was Ϸ67-71%. For clarity this level was set to 100% for each TnT mutant to determine the percentage of force activation and inhibition following the treatment. For two of the FHC TnT mutants, In16 and F110I, a residual Ca 2ϩ sensitivity of force was observed following the TnT(F110) and TnT(In16) treatment. The other FHC TnT mutants, I79N, R92Q, E163K, R278C, and wild-type HCTnT were able to replace the entire Tn complex within the time of incubation as judged by the complete loss of the Ca 2ϩ dependence of force. When Tn-displaced fibers were incubated with a preformed human cardiac TnI⅐TnC complex, dissolved in the relaxing solution (pCa 8), they underwent a gradual relaxation which was monitored by the inhibition of force in the absence of Ca 2ϩ (pCa 8). Incubation of the Tn-displaced fibers with the HCTnI⅐HCTnC complex restored the entire Tn complex, and the Ca 2ϩ regulation of force. Fig. 5 demonstrates the SDS-15% PAGE of representative Tn-displaced cardiac muscle preparations utilizing either the HCWTnT or the TnT(R278C) mutant. As shown, the TnT-treated fibers had a greatly reduced endogenous TnI⅐TnC content (lanes 2 and 7) which was completely reconstituted with a preformed HCTnI⅐HCTnC complex ( lanes  3 and 8). The stoichiometric ratio of other muscle proteins in the fibers: F-actin, tropomyosin, myosin light chains 1 (ELC), and 2 (RLC), was not changed following the TnT treatment/ reconstitution. A Western blot of the TnT(R278C)-treated and HCTnI⅐HCTnC-reconstituted fibers performed with the TnT (A) and TnI (B) specific antibodies (Goat anti-HCTnT polyclonal and monoclonal mouse anti-TnI IE7 GAM-PO) is presented in Fig. 6. As shown, the TnT(R278C)-treated fibers lacked the endogenous TnI that was displaced (lane 1) during the treatment and was easily reconstituted with human cardiac TnI during incubation of the TnT(R278C)-treated fibers with the recombinant HCTnI⅐HCTnC complex (lane 2).
Steady-state Force Development- Fig. 7 summarizes the effect of the FHC TnT mutants on the relaxation (pCa 8) and force recovery (pCa 4) in skinned cardiac muscle preparations following the Tn displacement and reconstitution procedures. The dashed line indicates the level of Ca 2ϩ -unregulated force following the TnT treatment (set to 100% for each TnT mutant). The open bars represent the force developed in the presence of Ca 2ϩ , while the dashed bars indicate the level of force in the absence of Ca 2ϩ (Fig. 7). As shown, the level of force inhibition (pCa 8) and force activation (pCa 4) following the TnT treatment and HCTnI⅐HCTnC reconstitution depended on the FHC TnT mutant used in the Tn-displacement procedure. The fibers treated with HCWTnT and reconstituted with HCTnI⅐HCTnC inhibited (ϪCa 2ϩ ) Ϸ95.5 Ϯ 7.8% of the Ca 2ϩunregulated force (100%) and after being switched to the high Ca 2ϩ solution (pCa 4) developed Ϸ90.6 Ϯ 6.7% of the force. The FHC TnT mutants, I79N and E163K, inhibited and activated force in a similar manner to HCWTnT (Fig. 7). Slightly impaired inhibition of force (in the absence of Ca 2ϩ ) was demonstrated for R92Q, F110I, and R278C, whereas In16 only inhibited Ϸ74.7 Ϯ 8.0% of the force. Activation of force (in the presence of Ca 2ϩ ) was also very impaired for In16 (35 Ϯ 3%), while R278C and F110I-treated fibers developed Ϸ79 Ϯ 10.4 and 80 Ϯ 12.3% of force, respectively (Fig. 7).
The Ca 2ϩ Sensitivity of Force Development-Skinned porcine muscle fibers reconstituted with human cardiac Tn were tested for their Ca 2ϩ sensitivity of force development (Fig. 8). As shown in Fig. 8A, porcine fibers reconstituted with human cardiac Tn (T, I, and C) were less sensitive to Ca 2ϩ (⌬pCa 50 Ϸ Ϫ0. 19) than intact untreated fibers. However, when the porcine fibers were reconstituted with porcine Tn (T, I, and C), the same sensitivity of force development, before and after the treatment, was observed (data not shown). Thus, the Ϫ0.19 difference in the Ca 2ϩ sensitivity between the porcine fibers and those reconstituted with human cardiac Tn (Fig. 8A) was due to the different Ca 2ϩ response to the pig and human Tn complexes reconstituted into the skinned porcine muscle fibers. Therefore, the Ca 2ϩ dependence for all the FHC TnT mutants were compared with the Ca 2ϩ dependence of HCWTnT-treated, and HCTnI⅐HCTnC-reconstituted fibers (Fig. 8, B and C, Table  I). Because of the very low level of force recovery following the TnT(In16) treatment (35 Ϯ 3%), the Ca 2ϩ dependence of force development was not determined. Fig. 8B presents the Ca 2ϩ response of force of the TnT(I79N)-treated fibers. A slight increase in Ca 2ϩ sensitivity (⌬pCa 50 Ϸ ϩ0.15) was observed compared with the HCWTnT-treated fibers. A much larger change in the Ca 2ϩ sensitivity of force was observed for TnT(F110I)-and TnT(R278C)-treated fibers. These two mutants caused the most dramatic increase (⌬pCa 50 Ϸ ϩ0.37 and Ϸ ϩ0.34, respectively) in the Ca 2ϩ sensitivity among all studied TnT proteins (Fig. 8C, Table I). Table I summarizes the pCa 50 values of the force-pCa relationship and the Hill coefficients for the fibers treated with wild-type HCTnT and the FHC TnT mutants. As shown, the TnT(I79N) and TnT(R92Q) increased the Ca 2ϩ dependence of force development by ⌬pCa 50 Ϸ ϩ0.15 and Ϸ ϩ0.18, respectively, compared with HCWTnT and their Hill coefficients were somewhat lower than that for the HCWTnT-treated fibers. As mentioned above, the effect of TnT(R278C) was the second largest among all of the TnT mutants (Table I, Fig. 8C). However, the cooperativity parameters (Hill coefficients) of the force-pCa dependence for TnT(R278C)-and TnT(F110I)-treated fibers were significantly lower than that of the HCWTnT-treated fibers ( Table I). The TnT(E163K) mutant was similar to HCWTnT when reconstituted in skinned porcine fibers, with a ⌬pCa 50 Ϸ ϩ0.07 compared with the HCWTnT-treated fibers (Table I). DISCUSSION Our results indicate that the FHC TnT mutations play an important role in the Ca 2ϩ regulation of ATPase/force development and the activation and inhibition of ATPase/force. The TnT(R278C)-and TnT(F110I)-reconstituted fibers demonstrated dramatically increased Ca 2ϩ sensitivity of force, while TnT(R92Q)-and TnT(I79N)-treated fibers showed a moderate increase (Table I). The deletion mutant, TnT(In16), significantly decreased both the activation and the inhibition of force, and substantially decreased the activation and the inhibition of actin-Tm-activated myosin-ATPase activity when reconstituted in thin filaments. The ATPase activation was also impaired by TnT(F110I), while the inhibition was reduced by TnT(R278C). The TnT(E163K) mutation had the smallest effect on the Ca 2ϩ sensitivity of force; however, it caused an elevated activation of the ATPase activity in reconstituted thin filaments. These observed changes in the Ca 2ϩ regulation of the ATPase and force development could be a clue to understanding the altered cardiac contractility seen in humans with these mutations (11,13,26). To distinguish how any particular TnT mutation could cause these alterations, a fundamental  knowledge of the role of TnT in the Ca 2ϩ regulation of cardiac muscle contraction is essential. The role of the regions containing these mutations is also critical.
TnT anchors the TnI⅐TnC complex to the thin filaments through an interaction with TnI and tropomyosin, and also confers Ca 2ϩ sensitivity to actomyosin-ATPase activity when complexed with TnI, TnC, and Tm (4 -8). Studies with proteolytic fragments of TnT have indicated that the functionally important sites of TnT are mostly located in the COOH-terminal half of the molecule. This region of TnT interacts with Tm, TnC, TnI, and actin (7,(47)(48)(49)(50). Two classes of interactions have been proposed to occur within the COOH-terminal domain of TnT. 1) Structural, Ca 2ϩ -independent interactions, between the COOH-terminal domain of TnC and the NH 2 -terminal domain of TnI; and 2) the regulatory, Ca 2ϩ -dependent interactions between the NH 2 -terminal, Ca 2ϩ -specific domain of TnC, and the COOH-terminal domain of TnI (containing the inhibitory region) (7). The dependence of the Ca 2ϩ -sensitizing function of TnT on the amino acid sequence of its NH 2 terminus remains an open question (8,51). The large number of TnT isoforms, compared with other thin filament proteins suggests the importance of the NH 2 -terminal variable region of TnT in the Ca 2ϩ regulation of skeletal muscle contraction.
The FHC TnT mutations studied in this paper have also been investigated by others. Several studies utilizing various techniques, including in vitro motility assays, expression of different TnT isoforms (human, rat, embryonic, and adult) in different systems such as quail myotubes, rat cardiac myocytes, feline cardiac myocytes, transgenic animal models etc., have been reported during the past few years. Some of them gave conflicting results, perhaps due to the utilization of different systems. Lin et al. (31), using recombinant rat cardiac TnT, containing a mutation in an equivalent position to the TnT(I79N) mutation, showed that troponin, containing this TnT, had normal function in terms of its affinity for Tm, Tn induced binding of Tm to actin, cooperative binding of myosin S1 to thin filaments and the Ca 2ϩ sensitivity of acto-S1 ATPase activity. They found, however, that regulated thin filaments, containing this Tn, moved 50% faster over HMM in an in vitro motility assay than control filaments and suggested that this could possibly lead to altered contractility in cardiac muscle. Sweeney et al. (33) reported that TnT(I79N)-and TnT(R92Q)transfected quail myotubes demonstrated decreased Ca 2ϩ sensitivity of force production, whereas the unloaded shortening velocity was increased about 2-fold. In a recent paper of Rust et al. (35), an embryonic isoform of rat TnT and two FHC TnT mutations, TnT(I79N) and TnT(R92Q) made from it, were expressed in adult rat cardiac myocytes. Measurements of isometric force in these myocytes demonstrated significantly decreased Ca 2ϩ sensitivity with unaltered maximum tension. Marian et al. (23) expressed human cardiac TnT containing the R92Q mutation in feline cardiac myocytes and found that their contractility was impaired. A recent paper of Yu et al. (27) demonstrated normal myofibrillar formation and sarcomere assembly when the R92Q mutation was expressed in adult rabbit myocardium. In a report from Leinwand's lab (25), transgenic mouse lines containing different amounts of mouse cardiac TnT with the In16 mutation were studied. These animals exhibited sarcomeric disarray and had significant diastolic dysfunction. Animals with higher levels of transgene expression died within 24 h of birth. An earlier report from Watkins et al. (21), utilized wild-type and the same truncated mutant HCTnT to transfect quail myotubes and found that Ca 2ϩ -activated force was significantly reduced with the mutant HCTnT compared with wild-type HCTnT.
Our results with the truncated TnT(In16) mutant are in agreement with those reported above. The level of force development in TnT(In16)-treated and HCTnI⅐HCTnC-reconstituted fibers was very low in comparison to HCWTnT or other FHC TnT mutants. The relaxation in these fibers was also significantly impaired. Interestingly, these perturbations in force development occurred even though the mutant did not bind well to the fibers and was not efficient in displacing the Tn complex. One could speculate that this mutation could result in altered stoichiometry of the thin filament proteins and lead to dysfunctional interactions between the thick and thin filaments. It is also possible that truncation of the COOH terminus of TnT caused by this mutation may change Ca 2ϩ -dependent interactions between TnT and the TnC⅐TnI complex and would ultimately result in reduced activation and relaxation during muscle contraction.
Our results are also in agreement with Morimoto et al. (32), who have reported that TnT(I79N) and TnT(R92Q) when reconstituted in skinned rabbit trabeculae, did not impair maximal force, but increased the Ca 2ϩ sensitivity of contraction. In a very recent study of Yanaga et al. (34), TnT(I79N), TnT(R92Q), as well as TnT(F110I), TnT(E244D), and TnT(R278C) were reconstituted in rabbit cardiac myofibrils and the Ca 2ϩ sensitivity of myofibrillar ATPase was measured. In concert with our results, the TnT(R278C) mutant caused the most substantial increase in Ca 2ϩ sensitivity of the myofibrillar ATPase, while TnT(I79N) and TnT(R92Q) produced about half of this effect. In contrast to their results with TnT(F110I), which had no effect on the Ca 2ϩ sensitivity of myofibrillar ATPase, our data indicated a significant increase in the Ca 2ϩ dependence of force development. Taking into consideration an impaired endogenous Tn displacement by TnT(F110I), one could speculate that the observed increase in the Ca 2ϩ sensitivity of force development was due to the increased sensitivity of porcine cardiac muscle preparations. However, the pCa 50 of the untreated porcine fibers was only 5.67, while that of TnT(F110I)-treated was as high as 5.85. Therefore, the observed increase in Ca 2ϩ sensitivity of force was primarily caused by the TnT(F110I) mutation. Possibly the results of Yanaga et al. (34) were affected by the reduced efficiency of TnT(F110I) to displace Tn in myofibrils. Stoichiometric reconstitution in thin filaments omits these problems, and as we demonstrated above, the actin-Tm-activated myosin-ATPase activity was impaired (73% of HCWTnT) by TnT(F110), while in their study an elevated myofibrillar ATPase was observed.
The effect of the TnT(R278C) on the Ca 2ϩ sensitivity of force is quite understandable since the mutation site is located in the TnT region which is known to interact with TnI, TnC, and Tm in a Ca 2ϩ -dependent manner (7,30,49,50). The change of a positively charged Arg to a hydrophobic Cys at position 278 could alter the Ca 2ϩ -dependent interactions between the COOH terminus of TnT and TnC, either directly or through interactions with TnI. The mutation might also alter the Ca 2ϩsensitive interaction of the COOH terminus of TnT and Tm. Interestingly, an Arg at position 278 is highly conserved among other cardiac TnT isoforms, however, most of the slow and fast skeletal muscle isoforms of TnT contain an Ala residue at this position. In the other TnT(F110I) mutant, which also produced an increase in the Ca 2ϩ sensitivity of force, the aromatic Phe residue is replaced with an aliphatic Ile. This change could be significant since this residue is highly conserved among all TnT isoforms, across both species and tissues. The effect of TnT(F110I) on the Ca 2ϩ sensitivity of force development is quite surprising since this region of TnT (exon 10) is thought to interact with tropomyosin in a Ca 2ϩ -independent manner. Perhaps an allosteric effect of this mutation on the COOH-terminal domain of TnT, which interacts with the TnI⅐TnC complex in a Ca 2ϩ -dependent manner, occurs here. On the other hand, as determined by the displacement experiments, the F110I mutation most probably results from a decreased affinity of the TnT for Tm. This could facilitate the interaction of the COOH terminus of TnT, with TnI and TnC in a Ca 2ϩ -dependent manner. Two other FHC mutations, TnT(I79N) and TnT(R92Q), are also located in a TnT-Tm interaction interface and are not thought to be regulated by Ca 2ϩ . One could speculate that these mutations might affect interaction of TnT with Tm and possibly alter the movement of Tm on actin. These alterations could in turn affect the interaction of actin-Tm with the myosin heads and influence the contractility and Ca 2ϩ sensitivity of cardiac muscle.
TnT(E163K) had the smallest effect on the Ca 2ϩ sensitivity of force, however, when reconstituted into thin filaments, it caused the highest observed activation of actin-Tm activated myosin-ATPase activity. According to Malnic et al. (7), this residue is located in a region of cardiac TnT that contains intrinsic activation properties. The change of the negatively charged Glu to a positive Lys could possibly facilitate these properties. An elevated level of the ATPase activity observed with this mutation may produce an altered cross-bridge duty cycle and possibly alter the kinetics of actin-Tm-Tn-myosin interaction. This in turn could contribute to energetic perturbations during muscle contraction.
Dramatic increases in the Ca 2ϩ regulation of force development observed in our skinned fiber experiments could lead to the altered cardiac contractility seen in humans with these mutations. The increased (or decreased) Ca 2ϩ sensitivity of force that accompanies these mutations would reduce (or increase) the concentration of Ca 2ϩ required to produce the equivalent tension response to that seen in a normal muscle fiber. Since the troponin complex is a significant buffer within the cardiac myocyte, any alteration in its Ca 2ϩ affinity would be expected to alter overall cellular Ca 2ϩ homeostasis. These alterations might also be expected to change the Ca 2ϩ transient (magnitude and time course) and thereby any of the multiple Ca 2ϩ -dependent processes within the cell, e.g. SR function, calmodulin-regulated systems, ion channels, etc. The changes in Ca 2ϩ sensitivity with and/or without changes in the maximum level of ATPase activity or force may contribute to an impaired inotropic response. These abnormalities could lead to major alterations in critical cell signaling events and ultimately to catastrophic results, e.g, arrhythmia, sudden death, etc. Moreover, the changes in Ca 2ϩ homeostasis could result in altered expression of a variety of genes including stress response and hypertrophy related genes that could be themselves causal for FHC. Further studies will be necessary to fully understand the mechanisms involved in the production of FHC caused by these mutations in TnT.