Characterization of troponin T dilated cardiomyopathy mutations in the fetal troponin isoform.

The major goal of this study was to elucidate how troponin T (TnT) dilated cardiomyopathy (DCM) mutations in fetal TnT and fetal troponin affect the functional properties of the fetal heart that lead to infantile cardiomyopathy. The DCM mutations R141W and DeltaK210 were created in the TnT1 isoform, the primary isoform of cardiac TnT in the embryonic heart. In addition to a different TnT isoform, a different troponin I (TnI) isoform, slow skeletal TnI (ssTnI), is the dominant isoform in the embryonic heart. In skinned fiber studies, TnT1-wild-type (WT)-treated fibers reconstituted with cardiac TnI.troponin C (TnC) or ssTnI.TnC significantly increased Ca(2+) sensitivity of force development when compared with TnT3-WT-treated fibers at both pH 7.0 and pH 6.5. Porcine cardiac fibers treated with TnT1 that contained the DCM mutations (R141W and DeltaK210), when reconstituted with either cardiac TnI.TnC or ssTnI.TnC, significantly decreased Ca(2+) sensitivity of force development compared with TnT1-WT at both pH values. The R141W mutation, which showed no significant change in the Ca(2+) sensitivity of force development in the TnT3 isoform, caused a significant decrease in the TnT1 isoform. The DeltaK210 mutation caused a greater decrease in Ca(2+) sensitivity and maximal isometric force development compared with the R141W mutation in both the fetal and adult TnT isoforms. When complexed with cardiac TnI.TnC or ssTnI.TnC, both TnT1 DCM mutations strongly decreased maximal actomyosin ATPase activity as compared with TnT1-WT. Our results suggest that a decrease in maximal actomyosin ATPase activity in conjunction with decreased Ca(2+) sensitivity of force development may cause a severe DCM phenotype in infants with the mutations.

The major goal of this study was to elucidate how troponin T (TnT) dilated cardiomyopathy (DCM) mutations in fetal TnT and fetal troponin affect the functional properties of the fetal heart that lead to infantile cardiomyopathy. The DCM mutations R141W and ⌬K210 were created in the TnT1 isoform, the primary isoform of cardiac TnT in the embryonic heart. In addition to a different TnT isoform, a different troponin I (TnI) isoform, slow skeletal TnI (ssTnI), is the dominant isoform in the embryonic heart. In skinned fiber studies, TnT1wild-type (WT)-treated fibers reconstituted with cardiac TnI⅐troponin C (TnC) or ssTnI⅐TnC significantly increased Ca 2؉ sensitivity of force development when compared with TnT3-WT-treated fibers at both pH 7.0 and pH 6.5. Porcine cardiac fibers treated with TnT1 that contained the DCM mutations (R141W and ⌬K210), when reconstituted with either cardiac TnI⅐TnC or ssTnI⅐TnC, significantly decreased Ca 2؉ sensitivity of force development compared with TnT1-WT at both pH values. The R141W mutation, which showed no significant change in the Ca 2؉ sensitivity of force development in the TnT3 isoform, caused a significant decrease in the TnT1 isoform. The ⌬K210 mutation caused a greater decrease in Ca 2؉ sensitivity and maximal isometric force development compared with the R141W mutation in both the fetal and adult TnT isoforms. When complexed with cardiac TnI⅐TnC or ssTnI⅐TnC, both TnT1 DCM mutations strongly decreased maximal actomyosin ATPase activity as compared with TnT1-WT. Our results suggest that a decrease in maximal actomyosin ATPase activity in conjunction with decreased Ca 2؉ sensitivity of force development may cause a severe DCM phenotype in infants with the mutations.
Troponin T (TnT) 1 isoforms are encoded by distinct genes in different muscle types: fast skeletal, slow skeletal, and cardiac muscle (1). Multiple isoforms of TnT have been identified in skeletal and cardiac muscle. The expression of more than one cardiac TnT isoform was first identified in chicken. Subsequently, multiple cardiac TnT isoforms were found in different species including the rabbit, rat, mouse, bovine, and human heart, and the number of isoforms expressed varies among different species. In the human heart, alternative splicing of exons 4 and 5 generates up to four different isoforms (TnT1-TnT4) with varying electrophoretic mobility. The four isoforms differ by the presence or absence of exons 4 and 5. Cardiac TnT1 has both exons 4 and 5, in cardiac TnT2 exon 4 is missing, in cardiac TnT3 exon 5 is missing, and in cardiac TnT4 both exons 4 and 5 are missing (2). The multiple cardiac TnT isoforms are expressed in a developmentally regulated manner. Anderson et al. (3) showed that TnT3 is the dominant TnT isoform in the adult human heart, and TnT1 and TnT2 are the two isoforms present in the fetal heart, with TnT2 being present at low levels (4). The switch in the expression of these different isoforms is believed to be at least partially responsible for the different Ca 2ϩ sensitivity seen between the neonatal and adult cardiac muscle (5).
Dilated cardiomyopathy (DCM) characterized by increased left ventricular cavity dimensions and systolic dysfunction is caused by mutations in sarcomeric proteins including cardiac TnT, troponin I (TnI), troponin C (TnC), actin, ␤-myosin heavy chain, and ␣-tropomyosin (Tm) (6,7). Kamisago et al. (8) reported the first TnT mutation as the cause of DCM in two unrelated families. This mutation leads to a deletion of the amino acid lysine at position 210 in exon 13. None of the individuals affected with DCM exhibited ventricular hypertrophy, a hallmark feature of hypertrophic cardiomyopathy. In affected family members, the TnT-⌬K210 mutation caused early-onset ventricular dilation. It also caused a high incidence of sudden cardiac death in both infants and adults. Two children (age, 1 month and 8 months, respectively) who died suddenly had a clinical diagnosis of infantile cardiomyopathy. Li et al. (9) reported another novel TnT mutation, R141W, in a large family of 72 members. Fourteen living members of the family clinically manifested DCM, predominantly in the second decade of their life. Five children (between the ages of 1 and 15 years) in this family died of DCM. Also, 17 members of this family died before genotypic analysis was carried out, presumably due to DCM. It is not known how many out of these 17 members were children. Previously, we investigated the functional properties of TnT DCM mutations in the adult isoform of TnT (TnT3) (10). Because a different TnT isoform is present in the embryonic heart (TnT1) and because both these DCM mutations cause infantile cardiomyopathy, we created mutations in the TnT1 isoform to elucidate the behavior of the mutations in the fetal TnT isoform.
In addition to a different TnT isoform, a different TnI isoform is expressed in the embryonic heart. Both slow skeletal TnI (ssTnI) and cardiac TnI (cTnI) are expressed in the heart during development (11). In humans, only ssTnI is present during the 12th week of gestation (12). In the human heart, ssTnI and cTnI mRNA and protein are both expressed in the neonate, and only cTnI is expressed in the adult heart. However, ssTnI expression persists in the human ventricle well into the first year of life (13,14). Although ssTnI and cTnI serve the same role in the regulation of muscle contraction by inhibiting the actomyosin ATPase activity, their responses to Ca 2ϩ and H ϩ ions are markedly different (15). In order to understand the behavior of DCM mutants in fetal troponin, we made the mutations in fetal TnT (TnT1) and utilized ssTnI instead of cTnI in our assays. Because the isoforms used in the assays represent the troponin isoforms in the fetal heart, these studies were designed to reveal how DCM mutations affect the functional properties of troponin in the fetal heart.

Cloning of Cardiac Muscle Troponin T WT, Isoform, and Mutants
The cDNA for human adult cardiac troponin T (TnT3-WT) was previously cloned in our laboratory by reverse transcription-PCR using a template of total RNA from human myocardium and oligonucleotide primers specific for the 5Ј and 3Ј regions of the respective coding sequences (16). The TnT1 isoform, which is the primary TnT isoform in the embryonic heart, was constructed using TnT3-WT as the template. Two mutations, R141W and ⌬K210, were introduced in TnT1 by the method of sequential overlapping PCR using the following primers (17): TnT1-R141W, 5Ј-CCGAGCAGCAGCGCATCTGGAATGAGCGGGAG; TnT1-R141W, 3Ј-TTCTCCCGCTCATTCCAGATGCGCTGCTGCTC; TnT1-⌬K210, 5Ј-AGCGGGAAAAGAAGAAGATTCTGGCTGAGA; and TnT1-⌬K210, 3Ј-CCTCTCAGCCAGAATCTTCTTCTTTTCCCG.
Amplified DNA fragments were subcloned into the Nco-BamHI site of the pet-3d expression vector. Clones that contained the correct inserts were sequenced to confirm the presence of the mutation.

Expression and Purification of TnT-WT, Isoform, and Mutants
Recombinant TnT3-WT, TnT isoform (referred to as TnT1-WT), and TnT1-DCM mutants were expressed in BL21 bacterial cells. Standard methods were utilized for purification of various TnTs (18,19). Briefly, bacterially expressed TnT was passed through an S-Sepharose column and eluted with a linear gradient of 0 -600 mM KCl. The fractions containing TnT were loaded onto a fast flow Q-Sepharose column and eluted with a 0 -500 mM salt gradient. The purity of the protein was Ͼ90%. The proteins were further passed through a DE-52 column that gave Ͼ95% pure protein. The purity of the proteins was verified on SDS-PAGE after each purification step.

Purification of cTnI and ssTnI
The cDNAs for human cTnI and ssTnI were previously cloned in our laboratory by reverse transcription-PCR using a template of total RNA from human myocardium and oligonucleotide primers specific for the 5Ј and 3Ј regions of the respective coding sequences (20). Escherichia coli BL21 bacterial cells were transformed with pET-3d constructs containing cTnI or ssTnI.
The proteins were initially purified on an S-Sepharose column, and the purity of cTnI and ssTnI was determined by SDS-PAGE. Fractions containing cTnI or ssTnI were purified using a TnC affinity column and eluted with a linear gradient consisting of 2 mM CaCl 2 and 1 M urea and 3 mM EDTA and 6 M urea. The purity of the proteins was verified on SDS-PAGE.

Measurement of the Ca 2ϩ Dependence of Force Development
This experiment was used to measure the steady-state isometric force and the Ca 2ϩ sensitivity of force development at both pH 6.5 and pH 7.0. The experiment was performed after displacement of the endogenous Tn complex and replacement with either TnT3-WT, TnT1-WT, or TnT1 DCM mutants and reconstitution with the human cTnI⅐TnC or human ssTnI⅐TnC complex. This protocol is well established in our laboratory (2,16,20).
Cardiac Skinned Muscle Preparation-Porcine hearts obtained from the slaughterhouse were transported to the laboratory in an ice-cold oxygen-saturated solution containing 140 mM NaCl, 4 mM KCl, 1. This partially skinned preparation was then stored at Ϫ20°C in the same solution without Triton X-100 and dissected into small bundles (100 -150 m in diameter) before each experiment.
TnI⅐TnC Complex Formation-The cardiac TnC was mixed with either ssTnI or cTnI in a molar ratio of 1:1.25 and dialyzed in a solution containing 6 M urea, 20 mM MOPS, pH 7.0, 0.5 mM CaCl 2 , 1 M KCl, 1 mM EGTA, and 1 mM dithiothreitol. After overnight dialysis, the TnI⅐TnC complex was dialyzed in solution containing 20 mM MOPS, pH 7.0, 0.5 mM MgCl 2 , and 1 mM dithiothreitol and decreasing KCl concentrations (1, 0.7, 0.4, 0.2, and 0.1 M). The excess TnI that precipitated from the complex was removed by centrifugation. The TnI⅐TnC complex was run on SDS-PAGE to verify stoichiometry before storage at Ϫ70°C.
Measurement of the Ca 2ϩ Dependence of Force-Porcine muscle fiber bundles (a bundle of three to five fibers isolated from a batch of glycerinated fibers) were mounted on a force transducer and treated with the pCa 8.0 relaxing solution containing 1% Triton X-100 for ϳ1 h. The average length and diameter of the fibers selected for the experiment were ϳ1.2-1.5 mm and 120 -150 m. The composition of pCa 8.0 solution was 10 Ϫ8 M Ca 2ϩ , 1 mM Mg 2ϩ , 7 mM EGTA, 5 mM MgATP, 20 mM imidazole, pH 7.0, 20 mM creatinine phosphate, and 15 units/ml creatinine phosphokinase, ionic strength ϭ 150 mM. This permeabilizes the muscle membrane and allows us to gain access to the myofilament proteins. Subsequently, the fibers were transferred to pCa 8.0 solution without Triton X-100 and then into pCa 4.0 solution for the initial force determination. The composition of the pCa 4.0 solution is the same as that of the pCa 8.0 solution, except that 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 8.0 to pCa 4.0. To displace the endogenous Tn complex, the fibers 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 ϳ0.8 -1 mg/ml TnT3-WT, TnT isoform, or TnT DCM mutants for 1 h at room temperature. After a 1-h incubation, the fibers were incubated in fresh TnT protein for another hour. This was done to ensure the displacement of endogenous troponin from the fibers. Displaced fibers were then washed with the same solution without the protein (10 min at room temperature) and tested for the Ca 2ϩ -unregulated force that developed due to the absence of the endogenous porcine cTnI and TnC. The Ca 2ϩ regulation of steady-state force was restored with a preformed human cTnI⅐TnC complex. The reconstitution with the TnI⅐TnC complex (30 M) was performed in the pCa 8.0 solution for ϳ1.5 h at room temperature for the force to reach a stable level. Control fibers were run in parallel and treated with the same solutions without the proteins. The final Ca 2ϩ sensitivity of force development was determined after human cTnI⅐TnC reconstitution as described before by exposing the fiber to increasing Ca 2ϩ concentration from pCa 8.0 to pCa 4.0. The data were analyzed using the Hill equation in Sigma plot. The Hill equation is written as follows: % relative force ϭ 100 ϫ [Ca 2ϩ ] nH / ([Ca 2ϩ ] nH ϩ pCa 50 nH ), where pCa 50 determines Ca 2ϩ concentration of the solution in which 50% of force is produced, and n H is the Hill coefficient.

Reconstituted Actomyosin ATPase Assay
The native proteins used are rabbit skeletal actin, porcine cardiac myosin, and porcine cardiac tropomyosin. Rabbit skeletal F-actin was prepared as described by Strzelecka-Golaszewska et al. (21). Porcine cardiac myosin was purified as described by Murakami et al. (22). Porcine cardiac Tm was prepared from pig ventricles according to Potter (23). Recombinant TnT, TnI, and TnC were used to make functional troponin complexes. The ATPase assays were performed with rabbit skeletal muscle F-actin (3.5 M) containing porcine cardiac Tm (1 M) and pre-formed Tn complexes (1 M) as described previously (2,16). 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, and 4 mM MgCl 2 , pH 7.0) were initiated with 2.5 mM ATP. After a 20-min incubation at 30°C, the reaction was terminated with 5% trichloroacetic acid. The inorganic phosphate that was released was measured according to the method of Fiske and SubbaRow (24).

Data Analysis
Statistical analysis of the differences between mean values was performed by Student's t test. Values are presented as mean Ϯ S.D.

Alterations in the Ca 2ϩ Sensitivity of Force Development
Caused by TnT1 DCM Mutations at pH 7.0 -In order to study the functional differences between TnT3 and TnT1, the endogenous porcine troponin complex was exchanged with either TnT3-WT or TnT1-WT and reconstituted with human cTnI⅐TnC, and the Ca 2ϩ sensitivity of contraction was measured at pH 7.0 and analyzed using the Hill equation. Before troponin exchange, the Ca 2ϩ dependence of force development was measured, and there was no statistically significant difference in either pCa 50 or Hill coefficient between the different fibers used (data not shown). As shown in Fig. 1A, porcine fibers treated with TnT1-WT increased Ca 2ϩ sensitivity of force development compared with TnT3-WT-treated fibers. This shift of ϩ0.12 pCa unit is consistent with previous results obtained from our laboratory (2) for the same proteins. Also, the Hill coefficient (an indicator of the cooperativity) significantly increased in TnT1 isoform-treated fibers. When porcine cardiac fibers were exchanged with TnT1-R141W and TnT1-⌬K210 (Fig. 1B), both of the DCM mutations decreased the Ca 2ϩ sensitivity of force development compared with TnT1-WT. The Ca 2ϩ unregulated force produced by fibers treated with TnT isoforms and mutants is shown in Table I. Although the ⌬K210 mutant had a lower Ca 2ϩ unregulated force than its respective TnT-WT, this difference was not statistically significant. Previous studies from our laboratory (10) showed that in the TnT3 isoform, only the TnT3-⌬K210 decreased Ca 2ϩ sensitivity of force development (and not TnT3-R141W) when com-pared with the TnT3-WT. However, in the TnT1 isoform, both mutations decreased the Ca 2ϩ sensitivity of force development, and the magnitude of the shift in Ca 2ϩ sensitivity was increased compared with what was observed in the TnT3 isoform. There was no significant difference in the Hill coefficient between TnT1-WT and TnT1 DCM mutant-treated fibers.
In the next series of experiments, porcine cardiac fibers were treated with either TnT3-WT, TnT1-WT, or TnT1 DCM mutants and reconstituted with ssTnI⅐TnC instead of cTnI⅐TnC. Results depicted in Fig. 1C show that the pCa 50 of TnT3-WTtreated fibers reconstituted with ssTnI⅐TnC is 5.55 Ϯ 0.02. However, when cTnI⅐TnC was used to reconstitute TnT3-WTtreated fibers, the pCa 50 observed was 5.39 Ϯ 0.02 (Fig. 1A). The increase in Ca 2ϩ sensitivity (ϩ0.16 pCa unit) observed upon ssTnI⅐TnC reconstitution is due to the well-known effect of ssTnI to lower the threshold for Ca 2ϩ activation of force development (25)(26)(27). The pCa-force relationship in fibers exchanged with TnT1-WT and reconstituted with ssTnI⅐TnC is also depicted in Fig. 1C. The Ca 2ϩ sensitivity of force development significantly increased in TnT1-WT-treated fibers compared with TnT3-WT-treated fibers reconstituted with ssTnI⅐TnC. Previous results show that (Fig. 1A) TnT1 isoform is more sensitive to Ca 2ϩ than TnT3 upon reconstitution with cTnI⅐TnC. Our results show that both the TnT1 isoform and ssTnI contribute to the additive increase in Ca 2ϩ sensitivity of force development in porcine cardiac fibers. We also measured the effect of the DCM mutations in the TnT1 isoform reconstituted with ssTnI⅐TnC in fiber studies. The pCa 50 value for fibers treated with TnT1-R141W and TnT1-⌬K210 and reconstituted with ssTnI⅐TnC was 5.46 Ϯ 0.03 and 5.47 Ϯ 0.02, respectively ( Fig. 1D; Table I). A significant decrease in Ca 2ϩ sensitivity was observed for both the TnT1 DCM mutants compared with TnT1-WT. Thus, the effect of TnT1-WT and ssTnI in increasing Ca 2ϩ sensitivity of force development is altered in the presence of TnT DCM mutations.
Ca 2ϩ Regulation of Steady-state Force Development with TnT1-DCM Mutants at pH 7.0 -In fiber studies, we also measured the isometric force produced under activating conditions (pCa 4.0). The maximal force recovered by fibers treated with TnT3-WT and reconstituted with cTnI⅐TnC was ϳ80%. The average rundown of the control fibers treated with buffers (without proteins) for the same time as the experimental fiber was 11-14%. Our results showed that the maximal force recovered by fibers exchanged with TnT1-WT and reconstituted with cTnI⅐TnC was significantly higher than fibers exchanged with TnT3-WT ( Fig. 2A; Table I).
The TnT1-R141W-treated fibers recovered 71% maximal force. Comparison of Ca 2ϩ activated maximal force in the fetal versus adult TnT isoform indicates that the R141W mutation behaves similar to the wild type in both the fetal and adult isoform. On the other hand, the ⌬K210 mutation decreased Ca 2ϩ activated maximal force in both the fetal and adult TnT isoforms (Table I). Fig. 2B illustrates the Ca 2ϩ activated maximal force achieved in fibers treated with TnT3-WT, TnT1-WT, and TnT1 DCM mutants and reconstituted with ssTnI⅐TnC. The maximal force recovered for fibers treated with TnT3-WT was ϳ85%. When the same TnT3-WT-treated fibers were reconstituted with cTnI⅐TnC, the maximal force recovered was 65% ( Fig. 2A). Therefore, our results indicate that ssTnI is able to markedly increase the amount of maximal force recovered, in addition to increasing the Ca 2ϩ sensitivity of force development. The maximal isometric force obtained for TnT1-WT-treated fibers reconstituted with ssTnI⅐TnC was 96%. This is a significant increase compared with 73% force recovered when TnT1-WTtreated fibers were reconstituted with cTnI⅐TnC ( Fig. 2A; Table  I). Our results indicate that the maximal recovered force is the highest when ssTnI is in combination with TnT1-WT rather than TnT3-WT. TnT1-R141W-treated fibers did not show significant changes in maximal force compared with TnT1-WT in the presence of ssTnI⅐TnC. However, in the ⌬K210 mutation-  treated fibers, we observed a significant decrease in the maximal force recovered (96% for TnT1-WT versus 70% for TnT1-⌬K210) when reconstituted with ssTnI⅐TnC.

Reconstituted Actomyosin ATPase Assays on TnT1 DCM Mutations-It is well known that TnI by itself inhibits ATPase activity and that the addition of TnC relieves this inhibition.
When TnT is added, there is an activation of actomyosin ATPase activity that is Ca 2ϩ -dependent (28). Fig. 3A illustrates the ATPase inhibition in the absence of Ca 2ϩ (ϪCa 2ϩ ) in Tn complexes containing cTnI⅐TnC. The actomyosin ATPase activity in the absence of human cardiac troponin complex was considered to be the basal activity. In the absence of Ca 2ϩ , ATPase was maximally inhibited at 2 M [Tn]. Tn complexes containing TnT3-WT were able to inhibit 86.4% of the basal activity. TnT1-WT-containing complexes were not able to inhibit as well as TnT3-WT (75 Ϯ 2% for TnT1-WT versus 86.4 Ϯ 2% for TnT3-WT). This difference in inhibition was found to be statistically significant by Student's t test (p Ͻ 0.001). Both DCM mutations R141W and ⌬K210 in the TnT1 isoform inhibited ATPase activity similar to TnT1-WT at the various Tn concentrations.
In order to understand how the DCM mutations would affect ATPase inhibition in the fetal heart at the level of troponin, we utilized ssTnI⅐TnC to make troponin complexes containing TnT3-WT, TnT1-WT, and TnT1 DCM mutants. In the absence of Ca 2ϩ , when increasing concentrations of Tn complexes are added, the basal activity starts to fall. At 2 M, TnT3-WT⅐ssTnI⅐TnC complex was able to inhibit 60.5% of the basal ATPase activity. When TnT3-WT was complexed with cTnI⅐TnC at the same concentration (2 M), 86.4% inhibition was achieved (Fig. 3A). TnT1-WT⅐ssTnI⅐TnC was able to inhibit only 48.5% of the basal ATPase activity. When complexed with cTnI⅐TnC, TnT1-WT was able to inhibit 75% of the basal ATPase activity (Fig. 3A). TnT1 DCM mutants complexed with ssTnI⅐TnC inhibited ATPase activity to a similar extent as TnT1-WT (ϳ49% for both proteins investigated). Our results show that complexes containing ssTnI⅐TnC are not able to inhibit as well as cTnI⅐TnC at all concentrations, and this difference was found to be statistically significant by Student's t test (p Ͻ 0.001). Fig. 4A illustrates ATPase activation in the presence of Ca 2ϩ (ϩCa 2ϩ ) at 1 M [Tn] (the concentration at which the maximal activity was achieved), and the results are indicated as a bar chart. The myosin ATPase activity in the absence of Tn was considered to be the basal activity (100% activity). Tn complex containing TnT3-WT and cTnI⅐TnC achieved ϳ160% activity. The ATPase activity of TnT1-WT was 157 Ϯ 6%. With respect to maximal ATPase activity, the fetal isoform of TnT, TnT1, behaves very similar to TnT3, which is the adult TnT isoform. The ATPase activity of TnT1-R141W was 140 Ϯ 2%, and the ATPase activity of TnT1-⌬K210 was 108 Ϯ 2%. The maximal ATPase activity of both DCM mutants in the TnT1 isoform was significantly decreased compared with the TnT1-WT. This result is similar to what was observed when the mutations were present in the adult cardiac TnT isoform (10). Fig. 4B depicts ATPase activity in the presence of Ca 2ϩ for TnT3-WT, TnT1-WT, and TnT1 DCM mutants complexed with ssTnI⅐TnC at 1 M Tn. The ATPase activity of TnT3-WT was ϳ157 Ϯ 3%, whereas TnT1-WT gave 172 Ϯ 5% activity. The increase in ATPase activity for TnT1-WT was found to be statistically significant (p Ͻ 0.01). The maximal ATPase activity of Tn complex containing TnT1-R141W was 143 Ϯ 4%, and the maximal ATPase activity of Tn complex containing TnT1-⌬K210 was 132 Ϯ 1% (p Ͻ 0.001). The maximal activity for the TnT1 DCM mutants was significantly lower compared with TnT1-WT (p Ͻ 0.001) when complexed with ssTnI⅐TnC. Our results suggest that ssTnI modulates both ATPase activation and inhibition.
Alterations in Ca 2ϩ Sensitivity of Force Development and Maximal Force Caused due to TnT1 DCM Mutations at pH 6.5- Fig. 5A illustrates the effect of acidic pH on TnT1-WT and TnT1 DCM mutants in the presence of cTnI. As can be seen, in TnT1-WT-treated fibers, acidic pH caused a decrease of 0.65 pCa unit (pCa 50 of 4.86 Ϯ 0.04 at pH 6.5 compared with pCa 50 of 5.51 Ϯ 0.03 at pH 7.0). It is well known that the pCa-force relationship shifts to the right under acidic pH conditions. Also at pH 6.5, a significant decrease in the Hill coefficient was observed (n H ϭ 1.44 at pH 6.5) in TnT1-WT-treated fibers. When TnT1-R141W and TnT1-⌬K210 mutations were used in the fiber studies, after reconstitution with cTnI⅐TnC, both the TnT1 DCM mutations caused a statistically significant decrease in Ca 2ϩ sensitivity of force development when compared with TnT1-WT-treated fibers at pH 6.5. The relative magnitude of decrease in Ca 2ϩ sensitivity of force development increased as the pH value was altered from 7.0 to 6.5 for both mutations (0.8 pCa unit for TnT1-R141W and 0.7 pCa unit for TnT1-⌬K210). The steady-state isometric force achieved with maximal calcium activation was lowered at pH 6.5 for TnT1-WT as well as for TnT1 DCM mutants (Fig. 6A). TnT1-WT-treated fibers recovered 57% of maximal force at pH 6.5 compared with 72% of maximal force at pH 7.0. The TnT1-R141W-treated fibers recovered 48.6% of the maximal force, and the TnT1-⌬K210-treated fibers restored only 30.3% of the initial force. Comparison of Ca 2ϩ activated maximal force at pH 6.5 in the fetal versus adult TnT isoform indicates that the R141W mutation does not alter the maximal force recovered in both the fetal and adult TnT isoform (Table II). However, the ⌬K210 mutation significantly decreased the maximal force in both the fetal and adult TnT isoforms (Table II).
We also investigated the pCa-force relationship under acid pH conditions in porcine fibers treated with TnT3-WT, TnT1-WT, and TnT1 DCM mutants and reconstituted with ssTnI⅐TnC. At pH 6.5, a pCa 50 value of 5.22 Ϯ 0.04 was obtained for TnT3-WT-treated fibers (Table II). At pH 7.0, TnT3-WT-treated fibers reconstituted with ssTnI⅐TnC gave a pCa 50 value of 5.55 Ϯ 0.02. The ⌬pCa 50 , which is the difference between the pCa 50 at pH 7.0 and pH 6.5, was equal to ϩ0.33 pCa unit. This shift in pCa 50 obtained in TnT3-WT-treated fibers with ssTnI⅐TnC reconstitution was significantly smaller than what was observed after cTnI⅐TnC reconstitution (⌬pCa 50 ϭ 0.58) . This is due to the effect of ssTnI, which can resist changes in pH better than cTnI. Results depicted in Fig.  5B illustrate that at pH 6.5, the pCa 50 of TnT1-WT-treated fibers reconstituted with ssTnI⅐TnC is 5.29 Ϯ 0.02. This small increase in pCa 50 observed for TnT1-WT compared with TnT3-WT was not statistically significant. When TnT1-R141W and TnT1-⌬K210 mutations were used in the fiber studies, both the TnT1 DCM mutations caused a statistically significant decrease in Ca 2ϩ sensitivity of force development when compared with TnT1-WT-treated fibers at pH 6.5 (Fig. 5B). No significant changes in Hill coefficient were observed between different fiber groups (Table II). Fig. 6B and Table II summarize the maximal force recovered at pH 6.5 for all groups of fibers reconstituted with ssTnI⅐TnC. The depression in maximal force normally induced by acid pH was significantly decreased upon ssTnI⅐TnC reconstitution in all fiber groups. DISCUSSION Mutations in cardiac TnT have been shown to cause both hypertrophic cardiomyopathy and DCM. The functional properties of TnT mutations causing hypertrophic cardiomyopathy and DCM have been previously characterized by several laboratories (10, 16, 29 -33). In all these studies, the mutations were created in the adult cardiac TnT isoform (TnT3). However, clinical data show that familial DCM caused due to TnT mutations affects infants as well as adults (8,9). Multiple cardiac TnT isoforms are expressed in the mammalian heart in a developmentally regulated manner. It is well established that TnT1 is the primary TnT isoform found in the fetal and neonatal heart (3,34). During perinatal development, expression of the TnT1 isoform decreases, and TnT3 expression increases and becomes the dominant TnT isoform. In order to elucidate the functional properties of DCM mutants at the level of fetal TnT, we created the TnT DCM mutations R141W and ⌬K210 in the primary fetal isoform of TnT (TnT1) and performed skinned fiber studies and reconstituted actomyosin ATPase assays. In addition to a different TnT isoform, a different TnI isoform is present in the fetal heart. The dominant TnI isoform in the fetal and neonatal heart is ssTnI. In order to understand how the TnT DCM mutations alter the functional properties of the troponin in the fetal heart, we utilized ssTnI instead of cTnI in our assays.
Studies on the Ca 2ϩ sensitivity of force development demonstrated that TnT1-WT-treated fibers reconstituted with cTnI⅐TnC caused a significant increase in Ca 2ϩ sensitivity and Hill coefficient of force development compared with TnT3-WTtreated fibers (Fig. 1A). This result is consistent with previous observations that TnT1 is more sensitive to Ca 2ϩ than TnT3 and suggests that the hypervariable region in the NH 2 terminus of cardiac TnT contributes to the Ca 2ϩ sensitivity of force development in the cardiac muscle (2,5,35). The R141W mutation showed a slight but not statistically significant decrease (Table I) in the Ca 2ϩ sensitivity of force development in the TnT3 isoform (10). However, in fetal troponin T (TnT1), the R141W mutation significantly decreased the Ca 2ϩ sensitivity of force development compared with TnT1-WT. The R141W mutation is located in a region of TnT that mainly interacts with Tm. Previous studies by Lu et al. (36) using a quartz crystal microbalance showed that the R141W mutation increased the affinity of cardiac TnT for tropomyosin. The authors postulated that the change in affinity of TnT for tropomyosin might amplify the inhibition of cTnI on the thin filament, necessitating more Ca 2ϩ binding to TnC to neutralize the TnI inhibition. The decrease in Ca 2ϩ sensitivity observed here suggests that the highly acidic NH 2 -terminal hypervariable region may modulate the interaction of TnT-R141W with Tm. Patients harboring the TnT-⌬K210 mutation show a high incidence of sudden cardiac death in infants (20% of affected individuals) (8). In fiber studies, TnT1-⌬K210 showed a significant decrease in Ca 2ϩ sensitivity of force development as well as maximal force.
When ssTnI⅐TnC was used to reconstitute TnT3-WT-treated fibers instead of cTnI⅐TnC, the Ca 2ϩ sensitivity of force development increased significantly. This result agrees with previous results that showed a differential response to Ca 2ϩ in myofilaments containing ssTnI compared with cTnI. Using adenovirus-mediated gene transfer, Westfall et al. (26) demonstrated that ectopic expression of ssTnI in isolated adult cardiac myocytes lowered the threshold for Ca 2ϩ activated tension development. The Ca 2ϩ sensitivity of force development increased even further in fibers treated with TnT1-WT and reconstituted with ssTnI⅐TnC, similar to what was reported by  Gomes et al. (37). This increase in Ca 2ϩ sensitivity is due to the contribution of both TnT1 and ssTnI. Both TnT1-R141W-and TnT1-⌬K210-treated fibers reconstituted with ssTnI⅐TnC decreased the Ca 2ϩ sensitivity compared with TnT1-WT-treated fibers. It is not known whether the TnT1 NH 2 -terminal hypervariable region alters the structure of TnT in the Ca 2ϩ -independent Tm binding region. A stronger interaction between cardiac TnC and ssTnI compared with cTnI would lower the threshold for Ca 2ϩ activated force development. It is possible that the interaction of ssTnI with TnC is altered in the presence of the TnT DCM mutations, leading to Ca 2ϩ desensitization of the thin filament. Also, the Ca 2ϩ activated maximal force increased significantly in fibers treated with TnT1-WT and reconstituted with ssTnI⅐TnC compared with TnT3-WTtreated fibers reconstituted with cTnI⅐cTnC. This could translate in vivo to enhanced cardiac contractility. However, TnT1-⌬K210-treated fibers showed a significant decrease in maximal force, and the TnT1-R141W-treated fibers compared with TnT1-WT-treated fibers reconstituted with ssTnI⅐TnC showed no change in maximal force. We also examined the ability of TnT1-WT and TnT1 DCM mutants to inhibit and activate actomyosin ATPase activity. In the absence of Ca 2ϩ , we found that Tn complexes containing TnT1-WT were unable to inhibit ATPase activity as well as TnT3-WT. This result is consistent with what was observed by Gomes et al. (2). Both the TnT1-R141W and ⌬K210 mutations were able to inhibit similar to TnT1-WT, indicating that the DCM mutations in the fetal TnT are not disrupting the inhibitory function of TnI. It is not clear whether the NH 2 terminus exerts a direct effect via interactions with Tm or acts indirectly through TnI and TnC. When Tn complexes containing ssTnI⅐TnC were utilized in ATPase inhibitory assays, we observed that Tn complexes containing ssTnI⅐TnC were not able to inhibit actomyosin ATPase activity as well as cTnI⅐TnC at all concentrations. The ability of ssTnI to significantly impair ATPase inhibitiory activity suggests that ssTnI possibly hinders the release of myosin head binding to the actin thin filament, and this would translate in vivo to incomplete diastolic relaxation. Interestingly, impaired diastolic relaxation was observed in transgenic mice overexpressing ssTnI. Again, in the presence of ssTnI⅐TnC, both mutations were able to inhibit as well as TnT1-WT.
No difference in maximal ATPase activation was observed between TnT3-WT and TnT1-WT complexed with cTnI⅐TnC (Fig. 4A). This result is consistent with what was observed by Gomes et al. (2). However, both DCM mutations in the TnT1 isoform caused a significant decrease in maximal ATPase activity (Fig. 4A). The importance of the NH 2 -terminal domain in the activation of actomyosin ATPase has been shown by Malnic et al. (38). Deletion of the NH 2 -terminal domain in TnT (residues 1-156 of chicken skeletal TnT) resulted in a significant reduction in the maximal ATPase activation (39). We also investigated maximal ATPase activity to determine whether ssTnI plays a major role in modulating the ATPase activity in the presence of Ca 2ϩ . The maximal ATPase activity of TnT3-WT complexed with ssTnI⅐TnC was similar to the activity obtained with cTnI⅐TnC complex. However, the maximal ATPase activity significantly increased for TnT1-WT and DCM mutants when complexed with ssTnI⅐TnC instead of cTnI⅐TnC. However, compared with TnT1-WT, the maximal activity for the TnT1 DCM mutants was significantly lower. We also measured the Ca 2ϩ sensitivity of force development and maximal force recovered at acid pH conditions (pH 6.5) for the TnT DCM mutations in the fetal isoform (TnT1) of TnT and fetal Tn (TnT1⅐ssTnI). Acidosis frequently occurs during birth, during which the intracellular pH varies from 6.2 to 7.4. When the pH was changed from 7 to 6.5, TnT3-WT-treated fibers showed a decrease in pCa 50 of 0.58 pCa unit. A rightward shift in pCa 50 was observed in TnT1-WT-treated fibers, but there was no significant difference in the Ca 2ϩ sensitivity of force development between TnT3-WT and TnT1-WT at pH 6.5 (Table II). At pH 6.5, in the TnT1 isoform, both the mutations significantly decreased the Ca 2ϩ sensitivity of force development similar to TnT1-WT, suggesting that the mutations are not resisting changes in pH. This result is expected because the R141W mutation is several residues away from the cluster of positively charged residues (residues 91-94 in TnT3) that are responsible for the determination of pH-dependent Ca 2ϩ regulation of cardiac muscle contraction (40).
It has been demonstrated by many laboratories that the change in pCa 50 and the force depression caused by acidic pH in neonatal heart are less than what is observed in the adult heart (26,41,42). The resistance to acidic pH is of great physiological significance because this could be one of the protection mechanisms for embryonic or neonatal heart against ischemia. We investigated the pCa-force relationship under acidic conditions (pH 6.5) in porcine fibers treated with TnT3-WT, TnT1-WT, and TnT1 DCM mutants and reconstituted with ssTnI⅐TnC. The shift in pCa 50 obtained with ssTnI⅐TnC reconstitution at pH 6.5 was significantly smaller than what was observed after cTnI⅐TnC reconstitution for all four proteins. This is due to the effect of ssTnI, which can resist changes in pH better than cTnI. It has been shown that the amino acid histidine at position 131 is primarily responsible for the ability of ssTnI to resist acidic pH (43). However, both the TnT1 DCM mutations caused a statistically significant decrease in Ca 2ϩ sensitivity of force development when compared with TnT1-WT-treated fibers at pH 6.5 (Fig. 5B). Table 2 summarizes the maximal force recovered at pH 6.5 for all groups of fibers reconstituted with ssTnI⅐TnC. The depression in maximal force normally induced by acid pH was significantly decreased upon ssTnI⅐TnC reconstitution in all fiber groups. A similar result was also observed by Wolska et al. (44) in transgenic mice overexpressing ssTnI in the heart. This suggests that the composition of the TnI isoform plays a major role in the determination of myocardial force development under acidic pH conditions. However, TnT1-R141W-treated fibers showed no change in maximal force compared with TnT1-WT-treated fibers reconstituted with ssTnI⅐TnC, whereas the TnT1-⌬K210 treated fibers showed a significant decrease in maximal force.
In summary, this study provides insight into the functional significance of DCM mutations in the fetal cardiac TnT isoform and fetal cardiac Tn. Our results demonstrate that both DCM mutations R141W and ⌬K210 decrease the Ca 2ϩ sensitivity of force development in both the TnT1 isoform and fetal Tn. Both mutations have in common a decrease in the Ca 2ϩ activated ATPase activity, with the decrease being more pronounced in ⌬K210 than in R141W in adult TnT (10), fetal TnT, and fetal Tn.
The in vitro results presented here suggest that both the R141W and ⌬K210 mutations would cause a severe DCM phenotype in the fetal and neonatal stage for the following reasons: the ⌬K210 mutation consistently decreased Ca 2ϩ sensitivity of force development, maximal force recovered, and maximal ATPase activity, irrespective of whether the mutation was present in the adult TnT, fetal TnT, or fetal Tn. The R141W mutation decreased Ca 2ϩ sensitivity of force development in the fetal TnT and fetal Tn. In the adult isoform of TnT, the R141W mutation caused no change in Ca 2ϩ sensitivity of force development compared with TnT3-WT. The TnT DCM mutations in the fetal Tn isoform showed significantly greater decreases in the Ca 2ϩ sensitivity of force development (compared with wild-type TnT1) relative to the decreases observed when these mutations were present in the adult TnT3 isoform. This greater decrease in change in Ca 2ϩ sensitivity in combination with a decrease in maximal actomyosin ATPase activity in fetal TnT1 may contribute to a more severe phenotype in children with these mutations, consistent with clinical data (8). In addition to TnT and TnI, actin and tropomyosin and myosin heavy chain also have different isoforms during cardiac development. How they affect the contribution of TnT and TnI to Ca 2ϩ sensitivity and ATPase activity remains to be established.