A Mutation in TNNC1-encoded Cardiac Troponin C, TNNC1-A31S, Predisposes to Hypertrophic Cardiomyopathy and Ventricular Fibrillation*

Background: Cardiac troponin C mutations are rare causes of HCM. A novel mutation in TNNC1 gene was identified in a pediatric HCM patient. Results: Functional characterization demonstrated increased myofilament Ca2+ affinity. Conclusion: The proband presented with ventricular fibrillation, aborted sudden cardiac death associated with myofilament dysregulation. Significance: The newly identified cardiac troponin C mutation predisposes to pathogenesis of a fatal arrhythmogenic subtype of HCM. Defined as clinically unexplained hypertrophy of the left ventricle, hypertrophic cardiomyopathy (HCM) is traditionally understood as a disease of the cardiac sarcomere. Mutations in TNNC1-encoded cardiac troponin C (cTnC) are a relatively rare cause of HCM. Here, we report clinical and functional characterization of a novel TNNC1 mutation, A31S, identified in a pediatric HCM proband with multiple episodes of ventricular fibrillation and aborted sudden cardiac death. Diagnosed at age 5, the proband is family history-negative for HCM or sudden cardiac death, suggesting a de novo mutation. TnC-extracted cardiac skinned fibers were reconstituted with the cTnC-A31S mutant, which increased Ca2+ sensitivity with no effect on the maximal contractile force generation. Reconstituted actomyosin ATPase assays with 50% cTnC-A31S:50% cTnC-WT demonstrated Ca2+ sensitivity that was intermediate between 100% cTnC-A31S and 100% cTnC-WT, whereas the mutant increased the activation of the actomyosin ATPase without affecting the inhibitory qualities of the ATPase. The secondary structure of the cTnC mutant was evaluated by circular dichroism, which did not indicate global changes in structure. Fluorescence studies demonstrated increased Ca2+ affinity in isolated cTnC, the troponin complex, thin filament, and to a lesser degree, thin filament with myosin subfragment 1. These results suggest that this mutation has a direct effect on the Ca2+ sensitivity of the myofilament, which may alter Ca2+ handling and contribute to the arrhythmogenesis observed in the proband. In summary, we report a novel mutation in the TNNC1 gene that is associated with HCM pathogenesis and may predispose to the pathogenesis of a fatal arrhythmogenic subtype of HCM.

the activation of the actomyosin ATPase without affecting the inhibitory qualities of the ATPase. The secondary structure of the cTnC mutant was evaluated by circular dichroism, which did not indicate global changes in structure. Fluorescence studies demonstrated increased Ca 2؉ affinity in isolated cTnC, the troponin complex, thin filament, and to a lesser degree, thin filament with myosin subfragment 1. These results suggest that this mutation has a direct effect on the Ca 2؉ sensitivity of the myofilament, which may alter Ca 2؉ handling and contribute to the arrhythmogenesis observed in the proband. In summary, we report a novel mutation in the TNNC1 gene that is associated with HCM pathogenesis and may predispose to the pathogenesis of a fatal arrhythmogenic subtype of HCM.
Hypertrophic cardiomyopathy (HCM), 7 defined as left ventricular hypertrophy without a clinically identifiable origin, affects ϳ1 of 500 individuals and is the most common cause of sudden death in the young athlete (1)(2)(3). Thought to be of genetic origin, HCM is inherited typically in an autosomal dominant fashion. Investigations over the past 20 years have led to the identification of hundreds of mutations associated with dozens of genes that have been linked to the pathogenesis of HCM (4,5). Principal among these are genes encoding components of the sarcomeric myofilament, which host the majority of HCM-associated mutations. Recently, mutations identified in TNNC1-encoded cardiac troponin C (cTnC), part of the sarcomeric thin filament, have been implicated as a rare cause of HCM (6 -8). Despite this progress, little is known about the arrhythmogenic role of TNNC1 mutations in the setting of car-diomyopathic disease. Furthermore, there is little mechanistic explanation for the increased risk of sudden cardiac death in some patients with this clinically heterogeneous disease (9).
Troponin C is part of the heterotrimeric regulatory troponin complex of the sarcomeric thin filament and serves as the Ca 2ϩ sensor of muscle contraction. The Ca 2ϩ -binding protein TnC works in concert with inhibitor troponin I (TnI) and troponin T (TnT), which provides a direct link to tropomyosin and assists in transducing the contractile signal to the rest of the thin filament. This process is initiated by the binding of cytosolic Ca 2ϩ during Ca 2ϩ -induced Ca 2ϩ release, the beginning of the Ca 2ϩ transient, which increases the binding affinity of TnC for TnI, thus pulling the TnI inhibitory domain away from its binding site on actin (10). The release of TnC allows the troponin-tropomyosin complex to move farther into the actin groove fully exposing the myosin binding sites on actin. The formation of active cross-bridges may then occur, allowing muscle tension to develop (10).
Cardiac TnC is a Ca 2ϩ -binding protein that belongs to the EF-hand superfamily which consists of two globular functional domains attached by a flexible linker (11). The N-domain is considered the regulatory domain and has one active Ca 2ϩ binding site (site II) that binds Ca 2ϩ with low affinity (ϳ10 5 M Ϫ1 ). The C-domain is known as the structural domain and contains two Ca 2ϩ /Mg 2ϩ binding sites (sites III and IV) that bind Ca 2ϩ at low concentrations (ϳ10 7 M Ϫ1 ) while also competitively binding Mg 2ϩ (ϳ10 3 M Ϫ1 ) (12). Therefore, only site II of the regulatory N-domain reversibly binds cytosolic Ca 2ϩ during cardiac contraction, and mutations that affect the function of cTnC may alter its global structure thus modifying its Ca 2ϩ affinity and/or interfering with protein-protein interactions necessary to appropriately transmit the Ca 2ϩ binding signal. Recently, we identified a novel TNNC1 mutation alanine substituted by serine at position 31 (A31S) in the non-functional Ca 2ϩ binding site I. This mutation may alter Ca 2ϩ binding to site II, which regulates the sensitivity of muscle contraction. Here, we investigated the source of the primary defect (increased Ca 2ϩ sensitivity in skinned muscles and ventricular tachycardia in the patient) that occurs in the presence of this mutation.
In addition to initiating cardiac contraction, cTnC is an important Ca 2ϩ buffer that assists in maintaining Ca 2ϩ homeostasis in the myocyte, and increased Ca 2ϩ binding affinity may result in arrhythmogenic Ca 2ϩ mishandling (13). Traditionally associated with mutations in cardiac ion channels, ventricular tachycardia has been identified in mouse models hosting Ca 2ϩ -sensitizing cTnT mutations (14). Furthermore, mouse models of a cTnI-R145G mutation have been shown to increase Ca 2ϩ sensitivity and prolong Ca 2ϩ transients (15). Despite these studies, the link between myofilament mutations and arrhythmogenesis, particularly in the context of HCM, remains relatively uncharacterized. To this end, we investigated the effects of a novel TNNC1 mutation identified in a host demonstrating youthful HCM presentation and repeated episodes of medically refractory ventricular fibrillation.

Study Control Cohorts
The absence of the mutation was confirmed in Ͼ26,600 reference alleles. Among these, were Ͼ800 unrelated, ethnically diverse, ostensibly healthy individuals recruited by Transgenomic Inc., 100 ostensibly health African American and 200 Caucasian American individuals from the Coriell Institute for Medical Research (Camden, NJ), and 200 additional Caucasian subjects with normal screening electro-and echocardiograms recruited from Olmsted County, Minnesota. Publically available databases from the Exome Chip Project (12,031 exomes), including the 1000 Genome Project (1,128 exomes) and NHLBI, National Institutes of Health Exome Sequencing Project (4,260 exomes), were also searched for the presence of the identified mutation (16).

Clinical Evaluation of TNNC1-A31S Proband
Clinical data were collected including pertinent personal and family history, physical examination, 12-lead electrocardiogram analysis, QT interval corrected for heart rate (QTc) measurement, and echocardiographic testing to determine mean left ventricular wall thickness, maximum left ventricular outflow tract gradient, and other parameters.

Functional and Structural Studies
Cloning, Expression, and Purification of Human Cardiac Troponin T, Troponin I, and Troponin C Mutants-The cTnI and cTnT cDNAs were cloned as previously described (17). The cTnC cDNA was cloned previously from total RNA obtained from human heart tissue. The sequential overlapping PCR method was used to introduce the A31S mutation into the cDNA (18). Standard methods previously used in this laboratory were utilized for the expression and purification of wildtype and mutant cTnC (19).
Fiber Preparation and Ca 2ϩ Dependence of Force Development Measurements-Fresh cardiac tissue was obtained from slaughterhouse pigs. Strips of papillary muscle 3-5 mm in diameter and ϳ5 mm in length were isolated from the left ventricle and skinned overnight in a 50% glycerol relaxing solution (10 Ϫ8 M [Ca 2ϩ ] free ) (6). Fibers were then transferred to a similar solution without Triton X-100 and stored at Ϫ20°C. Briefly, a skinned fiber bundle ϳ75-100 m in diameter was mounted using stainless steel clips to a force transducer and then immersed in a pCa 8.0 relaxation solution (conditions described in Ref. 6. The Ca 2ϩ dependence of force development was tested in skinned fibers at low, intermediate, and high concentration Ca 2ϩ solutions (pCa 8.0 -4.0) and calculated using the pCa calculator program developed in our laboratory (20). The native cTnC was depleted upon incubation of the fiber in a 1,2-cyclohexylenenitrilotetraacetic acid (CDTA) extracting solution (5 mM CDTA and 25 mM Tris (pH 8.4)) for ϳ 1.5 h. Fibers were considered extracted of cTnC when residual tension remaining in the fiber in pCa 4.0 was 15% or below. Fibers were then incubated with 28 M concentrations of mutant or WT cTnC diluted in pCa 8.0 for 1 h. The following equation was used to analyze data: % change in force ϭ 100 ϫ [Ca 2ϩ ] n / ([Ca 2ϩ ] n ϩ [Ca 2ϩ 50 ] n ), where [Ca 2ϩ 50 ] is the free [Ca 2ϩ ] that produces 50% force, and n Hill is the Hill coefficient. All fiber experiments were performed at room temperature.
Formation of Troponin Complexes-The purified individual troponin subunits including 2-(4Ј-iodoacetamidoanilino)naphthalene-6-sulfonic acid (IAANS)-labeled cTnC were first dialyzed against 3 M urea, 1 M KCl, 10 mM MOPS, 1 mM DTT, and 0.1 mM phenylmethanesulfonyl fluoride and then twice against the same buffer excluding urea. The protein concentrations of the individual subunits were determined using the Coomassie Plus kit and then mixed in a 1.3:1.3:1 cTnT:cTnI:cTnC molar ratio. After 1 h, the complexes were successively dialyzed against solutions containing decreasing concentrations of KCl (0.7, 0.5, 0.3, 0.1, 0.05, 0.025 M). Precipitated excess proteins during complex formation were removed by centrifugation. Proper stoichiometry was verified by SDS-PAGE before storing the troponin complexes at Ϫ80°C. Ternary troponin complexes were utilized in the actin-tropomyosin-activated myosin-ATPase assays containing the cTnC mutant.
Actin-Tropomyosin-activated Myosin-ATPase Assays; Minimum and Maximum ATPase-Porcine cardiac myosin, rabbit skeletal F-actin, and porcine cardiac tropomyosin were prepared as previously described (18). The protein concentrations used for actomyosin ATPase assays were: 0.6 M porcine cardiac myosin, 3.5 M rabbit skeletal F-actin, 1 M porcine cardiac tropomyosin, and 0 -2 M preformed troponin complexes as prepared above and were performed as single point assays that are linear over time (21). The proteins were in the following buffer conditions: myosin in 10 mM MOPS (pH 7.0), 400 mM KCl, 1 mM DTT; actin in 10 mM MOPS (pH 7.0), 40 mM KCl; tropomyosin in 10 mM MOPS (pH 7.0), 300 mM KCl, 1 mM DTT. The final ionic strength of the reactions was ϳ75 mM when considering the combined ionic contributions from all buffers. ATPase inhibition measurements were performed in a 0.1 ml reaction mixture: 3.4 mM MgCl 2 , 0.13 mM CaCl 2 , 1.5 mM EGTA, 3.5 mM ATP, 1 mM DTT, 11.5 mM MOPS (pH 7.0) at 25°C. The ATPase activation measurements were conducted using the same 0.1 ml buffer mixture with: 3.3 mM MgCl 2 and 1.7 mM CaCl 2 . ATP was added to initiate the reaction, which was quenched after 20 min using trichloroacetic acid at a final concentration of 35%. The precipitated assay proteins were removed by centrifugation. The amount of ATPase hydrolysis was determined by measuring the release of inorganic phosphate in the supernatant using methods established by Fiske and Subbarow (22).
Fluorescence Labeling of TNNC1-The TNNC1 was doublelabeled with IAANS at Cys-35 and Cys-84 and mono-labeled at Cys-84. IAANS was obtained from Molecular Probes, Plano, TX. Fluorescent labeling and purification of IAANS-labeled cTnC was performed according to established methods (23).
Determination of Apparent Ca 2ϩ Affinities by Fluorescence-IAANS-labeled cTnCs (WT and cTnC-A31S) were dialyzed into fluorescence buffer containing 2 mM EGTA, 5 mM Nitrilotriacetic Acid, 120 mM MOPS, 90 mM KCl. Before titration of isolated cTnC, 1.25 mM MgCl 2 and 1 mM freshly prepared DTT were added. For troponin complex formation and fluorescence experiments, fresh DTT was added before the titration because the dialysis buffer already contained 1.25 mM MgCl 2 that is needed for complex formation. Fluorescence measurements with isolated cTnC and the troponin complex were performed using double-labeled cTnC, with the IAANS label at Cys-35 and Cys-84. Steady state fluorescence measurements were performed using a Jasco 6500 spectrofluorimeter where IAANS fluorescence was excited at 330 nm, and emission was detected at 450 nm. The thin filaments were made according to Pinto et al. (24). The IAANS mono-labeling configuration was utilized for thin filament and thin filament ϩ subfragment 1 (S1) measurements, where Cys-35 in cTnC was mutated to Ser; therefore, only Cys-84 would be labeled. Putkey et al. (23) first described this method for Ca 2ϩ affinity measurements. The need for two labeling configurations, i.e. TnC labeled at both Cys-35 and -84 (double label) or with cTnC labeled only at Cys-84 (single label) is that in the presence of the other thin filament proteins, only one configuration responds to Ca 2ϩ . Therefore, we used the labeling configuration that provides the largest change in fluorescent signal at each given level of thin filament complexity (e.g. cTnC combined with the other troponin subunits (ternary complex) in the presence of tropomyosin and actin). The concentration of proteins used for fluorescence measurements was 10 M for isolated cTnC, 0.5 M for troponin, 0.05 mg/ml for thin filament, and 0.02 mg/ml for S1. This gives an overall stoichiometry between the thin filament and S1 as 1:1.58, respectively. We have previously determined that this is the ideal concentration/ratio of S1 that caused the half-maximal change in fluorescence from thin filaments (24). The change in the fluorescence spectra was recorded during the titration of microliter amounts of CaCl 2 . The concentration of free Ca 2ϩ and amounts of titrated Ca 2ϩ were obtained using the pCa calculator program (20). The program made corrections for dilution effects that occur during titration of Ca 2ϩ . The data were fit to a version of the Hill equation that accounted for the spectral changes that occur at a low Ca 2ϩ concentration and plotted using SigmaPlot 11.0. Circular Dichroism Measurements-Far UV circular dichroism spectra (CD) were collected using a 1-mm-path quartz cell SEPTEMBER 14, 2012 • VOLUME 287 • NUMBER 38 in a Jasco J-720 spectropolarimeter. Spectra were recorded at 195-250 nm with a bandwidth of 1 nm at a speed of 50 nm/min, whereas the resolution was 0.5 nm at room temperature. Ten scans were averaged, and numerical smoothing was not applied. The optical activity of the buffer was subtracted from relevant protein spectra. Mean residue ellipticity for the spectra was calculated utilizing the same Jasco system software using the following equation: [] MRE ϭ []/(10 ϫ Cr ϫ l), where [] is the measured ellipticity in millidegrees, Cr is the mean residue molar concentration, and l is the path length in cm (25). Protein concentrations were determined by the biuret reaction using bovine serum albumin as a standard. The CD experiments were performed using three different conditions: 1) apo state (divalent cation free) (1 mM EGTA, 20 mM MOPS, 100 mM KCl (pH 7.0)); 2) Mg 2ϩ -bound state (1 mM EGTA, 20 mM MOPS, 100 mM KCl, 2.075 mM MgCl 2 (pH 7.0)); 3) Ca 2ϩ -bound state (1 mM EGTA, 20 mM MOPS, 100 mM KCl, 2.075 mM MgCl 2 , 1.096 mM CaCl 2 (pH 7.0)). The experimental protein concentration for the WT and the A31S mutant cTnC was 0.2 mg/ml.

TNNC1-A31S in HCM and Ventricular Fibrillation
Three-dimensional Visualization-The cTnC-A31S mutation was visualized in the 1AJ4 Protein Data Bank file using PyMol software. PyMol is an open source molecular visualization program that allows manipulation of PDB files that contain molecular coordinates from x-ray crystallography-or nuclear magnetic resonance-based structures. The program allows mutagenesis of selected residues, portrays potential side chain interactions and potential for hydrogen bonding due to changes in the nature of and proximity of side chains.
Statistical Analysis-The experimental results were reported as x Ϯ S.E. and analyzed for significance using Student's t test at p Ͻ 0.05.

RESULTS
Genetic Analysis-Comprehensive HCM genetic analysis identified a heterozygous TNNC1 G 3 T mutation at nucleotide 91 resulting in a GCT 3 TCT alanine to serine alteration at residue 31 (TNNC1-A31S). This mutation was not identified in Ͼ26,600 reference alleles derived from ostensibly healthy individuals from a variety of racial and ethnic backgrounds. The proband did not host a compound mutation in eight other sarcomeric genes (MYH7, MYBPC3, MYL2, MYL3, TNNT2, TNNI3, TPM1, and ACTC) as well as three HCM phenocopyassociated genes (PRKAG2, GLA, and LAMP2).
Clinical Evaluation-The TNNC1-A31S mutation was identified in a Caucasian male who was symptom-free until his sentinel event of ventricular fibrillation at 3.75 years of age while sleeping at night. He arose, expressed concern to his parents, became syncopal, and was defibrillated successfully by paramedics with an automatic external defibrillator from ventricular fibrillation. He underwent intracardioverter defibrillator implantation and was maintained on ␤ blockade. Despite this therapy, he had five episodes of breakthrough ventricular fibrillation generally when emotionally excited and physically active, with single intracardioverter defibrillator shock restoring normal sinus rhythm in each case. He presented at age 5 to our institution for further evaluation. He had a negative family history for HCM and sudden cardiac death. Both parents, at 47 and 48 years of age, were negative for HCM by echocardiography (Fig. 1A). On echocardiographic examination, the proband demonstrated asymmetric septal wall hypertrophy with a mean left ventricular wall thickness of 20 mm (6 -8 mm normal range) with reverse curve morphology, an ejection fraction of 65%, diastolic dysfunction, and no left ventricular outflow tract obstruction (Fig. 1B). He had moderate left atrial enlargement. Electrocardiographic analysis demonstrated significant voltage criteria for biventricular hypertrophy, ST segment depression in anterior leads, and borderline QT prolongation with a QTc of 460 ms (Fig. 1C).
Cardiac Skinned Fiber Experiments-To assess whether the A31S mutation perturbs myofilament function, the Ca 2ϩ sensitivity and force recovery were evaluated using cTnC-depleted cardiac porcine fibers reconstituted with WT cTnC and cTnC-A31S. Incorporation of cTnC-A31S caused a leftward shift corresponding to an increase in Ca 2ϩ sensitivity of 0.17 pCa units with a pCa 50 of 5.63 in the WT to 5.80 in the mutant ( Fig. 2A and Table 1). The cooperativity of thin filament activation (n Hill ) ( Fig. 2A and Table 1) and force recovery % (P/P 0 ) (Fig. 2B) was unchanged.
Actomyosin ATPase Assays Using Reconstituted Troponin Complexes Containing cTnC-A31S-We next measured the ability of the troponin complex to activate or inhibit the actomyosin ATPase in the presence or absence of Ca 2ϩ , respectively. The activation of the ATPase (pCa 4) was measured with increasing amounts of preformed troponin complex (0 -2.0 M) (each point represents, n ϭ 6, performed in triplicate). The troponin complexes containing cTnC-A31S demonstrated increased thin filament activation compared with WT upon increasing concentrations of troponin. Specifically, there was an increase in the level of activation in the mutant cTnC-A31Sreconstituted thin filament (ϳ180%) compared with WT (ϳ150%) at 1.0, 1.5, and 2.0 M troponin (Fig. 3A). The inhibitory properties of the mutant cTnC-A31S complex was assessed at low Ca 2ϩ concentrations (pCa 8) by monitoring the ability to inhibit ATPase activity versus WT. The A31S mutant inhibited the actomyosin ATPase in a manner similar to WT although at higher concentrations of troponin (1.0 -2.0 M) (each point represents n ϭ 7, performed in triplicate). However, there was a statistically significant decrease in inhibition of the ATPase by the mutant troponin at the lower concentration range (ϳ52% at 0.3-0.8 M) compared with WT (ϳ40%) as shown in Fig. 3B. The Ca 2ϩ dependence of actomyosin ATPase activation was also evaluated, and the pCa 50 values were determined for the reconstituted thin filaments containing troponin complexes with either 100% WT, 100% cTnC-A31S, and 50:50 WT:cTnC-A31S (experiments performed n ϭ 8). When 100% mutant A31S troponin was incorporated into the reconstituted thin filaments, and Ca 2ϩ sensitivity was increased by ϩ0.38 pCa units, whereas when 50% of the mutant A31S troponin complex was utilized, Ca 2ϩ sensitivity increased by ϩ0.23 pCa units ( Fig.  3C and Table 1).

IAANS Fluorescence Measurements of cTnC Troponin, Thin Filament, and Thin Filament S1-containing cTnC-A31S-The
Ca 2ϩ affinity of isolated cTnC as measured by fluorescence obtained from IAANS, an extrinsic probe, indicated that the mutant cTnC-A31S (double label) had increased Ca 2ϩ affinity of ϩ0.17 pCa units compared with WT ( Fig. 4A and Table 2). When additional constituents (tropomyosin and actin) of the thin filament were included, there was a substantial increase in thin filament Ca 2ϩ affinity containing cTnC-A31S that increased ϩ0.56 pCa units compared with WT as shown in Fig.  4C and Table 2. In addition, spectral fluorescence changes from isolated cTnC were detected from the low affinity C-terminal Ca 2ϩ binding sites indicating that the probe picked up both sets  Table 1; B). Force recovery values (P) obtained after reconstitution with mutant and WT cTnC were compared with the level of force present before extraction of native cTnC (P 0 ) and were reported as % (P/P 0 ). Data are reported as the mean Ϯ S.E. (n ϭ 8 -10).  SEPTEMBER 14, 2012 • VOLUME 287 • NUMBER 38

JOURNAL OF BIOLOGICAL CHEMISTRY 31849
of binding events (Fig. 4A). Next, the Ca 2ϩ affinity of WT and cTnC-A31S was measured with the thin filament proteins (tropomyosin and actin) and myosin S1. Myosin S1 was added to each thin filament containing either WT or cTnC-A31S to determine whether the mutation further enhanced myofilament Ca 2ϩ sensitivity when strong cross-bridges formed between S1 and actin. Subsequently, we found that the cTnC-A31S mutant, in the absence of MgATP, increased the Ca 2ϩ affinity of the thin filament by ϩ0.25 compared with WT ( Fig.  4D and Table 2). In this way, the thin filament as well as crossbridge binding may play an important role in modulating the Ca 2ϩ sensitivity when the A31S mutation is present. Circular Dichroism of the HCM cTnC Mutant-To determine whether cTnC-A31S had an effect on the structure of the protein in the isolated state, the amount of secondary structure was determined in three conditions: apo (unbound), Mg 2ϩ -loaded, and Ca 2ϩ /Mg 2ϩ -loaded states (Fig. 5 and Table 3). Typically, the greatest change in ␣-helical content occurs when divalent cations, such as Mg 2ϩ , bind to the C terminus. Thus, the Mg 2ϩ -bound state is expected to accurately indicate structural changes occurring within the C terminus. The cardiac TnC-A31S mutation did not significantly change the ␣-helical content of the states examined, although the IAANS probe present in the N terminus was able to detect Ca 2ϩ binding events in the C terminus (Fig. 4A).

Mutations in Cardiomyopathic and Arrhythmic Disease-
Although some HCM-associated mutations have been associated with sudden death susceptibility based on survival studies of the kindred hosting the mutations, there is a paucity of mechanistic insight into the arrhythmogenic state caused by these mutations (26). Early studies identifying "malignant mutations" in families, such as (␤-myosin heavy chain) MYH7-R403Q and (cardiac troponin T) TNNT2-R92W, were associated with decreased Kaplan-Meyer survival when compared with families hosting so-called "benign" HCM mutations; however, detailed

TNNC1-A31S in HCM and Ventricular Fibrillation
mechanistic studies elucidating the sudden death susceptibility potentially caused by these mutations are needed (27,28).
Significant progress has been made in elucidating the genetic underpinnings of HCM; however, only a handful of mutations in TNNC1 have been identified. L29Q was one of the first mutations in TNNC1 to be associated with HCM (7). Although the effects of this mutation remain controversial, functional analysis has suggested that it may be a non-pathogenic variant based on inconsistent findings of altered Ca 2ϩ sensitivity (29 -32). We previously characterized four additional missense mutations, TNNC1-A8V, C84Y, E134D, and D145E, identified in a large cohort of unrelated patients with HCM (6,33). Cardiac fibers reconstituted with these HCM-associated cTnC mutants results in increased Ca 2ϩ sensitivity through mutation-specific alterations of dynamic interactions between cTnC and other components of the cardiac myofilament (33).
None of the earlier reported HCM TNNC1 mutations have been associated with arrhythmia, except that a recent study provides a link between the clinical outcome of sudden cardiac death and a TNNC1 mutation (8). A mutation in TNNC1, c.363dupG or p.Gln122AlafsX30 (truncation at position 122; cTnC-⌬122), has been identified in the proband age 19 in a small multi-generational family with HCM demonstrating reduced penetrance and variable expressivity (8). As with prior HCM-associated mutations, mechanistic studies identifying the pro-arrhythmic perturbation potentially imparted by this mutation have not been done to date (8). Therefore, we were unable to analyze shared functional properties between cTnC-A31S and the truncation mutant cTnC-⌬122 that may induce arrhythmia. The functional properties of this cTnC-⌬122 mutant are likely altered as it lacks Ca 2ϩ binding site IV, eliminating the coupling that exists between sites of the same   SEPTEMBER 14, 2012 • VOLUME 287 • NUMBER 38 domain and affecting the Ca 2ϩ buffering capacity of cTnC. Subsequently, the cTnC-A31S mutation located within the inactive Ca 2ϩ binding site I is unlikely to have restored Ca 2ϩ binding to any degree. However, in this case the A31S mutation may affect coupling between sites I and II of the same domain. Additional HCM-linked cTnC mutants may need to be identified and characterized to address how a cTnC mutant might cause arrhythmia.

TNNC1-A31S in HCM and Ventricular Fibrillation
In general, cardiomyopathic mutations that manifest significant physiological effects by an early age are considered most severe. In this study the patient presented significant cardiac symptoms involving ventricular fibrillation before the age of 4. In our previous study the TNNC1 mutation C84Y caused mild symptoms (syncope on exertion) at 8.4 years and was successfully managed by ␤-blockade (6). Comparison of clinical presentation of disease in patients bearing TNNC1 mutations with data measuring their functional consequences may provide additional insight.
Functional Properties of the HCM-associated cTnC-A31S Mutant-It has been hypothesized that a defining characteristic of TNNC1 HCM-associated mutations is the increased, or possibly equivalent, Ca 2ϩ sensitivity that may also alter the maximal myocyte force generation (34). To determine whether the mutant increased Ca 2ϩ sensitivity in a dominant-negative manner, we performed reconstituted actomyosin assays using 100% cTnC-A31S mutant and also in the "heterozygous" state using 50:50 cTnC-A31S:WT proteins to best represent the background of the patient. The intermediate pCa 50 obtained with 50% mutant cTnC (compared with 100% mutant or 100% WT) better-approximated the pCa 50 seen in the more intact skinned fiber system. This suggests that the mutation exerts its influence in a dominant negative manner, although the pCa 50 value may be altered by different amounts of cTnC mutant incorporation.
The reconstituted thin filaments containing cTnC-A31S displayed a significant increase in activation levels compared with those containing WT (Fig. 3A). Enhanced thin filament activation coupled with increased Ca 2ϩ sensitization of the myofilament may contribute toward the diastolic function seen in HCM patients. A likely scenario would be that the increase in Ca 2ϩ sensitivity heightens the contractile response and subsequently impairs the degree of relaxation achieved during diastole. Despite the increase in the actomyosin ATPase activation, the maximal force recovery was unchanged in skinned fibers. However, actomyosin ATPase inhibition measurements performed under relaxing conditions (pCa 8.5) showed that cTnC-A31S inhibited ATPase activity to the same degree as WT when (1.0 -2.0 M cTn) was added to the thin filament. This is consistent with bona fide HCM cTn mutations, which usually do not alter ATPase inhibition (33). The basal force in skinned fibers was unaffected (data not shown) (35). Taken together, the functional data support the HCM phenotype found in the patient.
The TNNC1-A31S Proband-In addition to left ventricular hypertrophy, the proband QTc is at the far upper limits of normal at 460 ms for a prepubertal boy. Usually associated with long QT syndrome, it has been previously demonstrated that elevated QTc is directly, albeit weakly, associated with the

TABLE 3 Summary of circular dichroism results for HCM cTnC mutants
The CD spectrum is reported for apo (no divalent cations bound) and Mg 2ϩ -bound and Ca 2ϩ /Mg 2ϩ -bound states, and error is reported as S.E. deg, degrees.

TNNC1-A31S in HCM and Ventricular Fibrillation
degree of left ventricular wall thickness (36,37). Although mutations in KCNQ1-encoded I Ks potassium channel, KCNH2encoded I Kr potassium channel, and SCN5A-encoded I Na sodium channel (NaV1.5, LQT3, gain-of-function), the three major genes associated with long QT syndrome, cannot be excluded from our genetic analysis, the elevated QTc observed in this proband is likely a reflection of profound hypertrophy rather than the presence of independent and concomitant long QT syndrome (38). The etiologies of ventricular fibrillation are diverse. In a structurally normal heart, purely electrical diseases can serve as a pathogenic substrate for ventricular fibrillation and sudden cardiac death. This includes channelopathic diseases such as long QT syndrome, Brugada syndrome, catecholaminergic polymorphic ventricular tachycardia, and short QT syndrome. The proband hosting the TNNC1 A31S mutation demonstrated concurrent hypertrophic remodeling of the heart; thus, it is more likely that the ventricular fibrillation and QT prolongation is secondary to his significant underlying hypertrophic cardiomyopathy.
Thin Filament Destabilization and HCM-Associated Alterations in Calcium-Affinity-Our identification of a missense mutation associated with both HCM and sudden cardiac death may represent a novel mechanism of thin filament sensitization. To gain mechanistic insight into how the cTnC-A31S mutation induces the myofilament dysfunction that underlies the presentation of HCM in the patient, we examined the manner in which the mutation altered the Ca 2ϩ affinity of the thin filament. These changes in Ca 2ϩ affinity likely involve alterations in the Ca 2ϩ binding capacity of cTnC that are translated into changes in Ca 2ϩ sensitivity within the myofilament. Previously, we found that although mutations occur within cTnC, they can impact the Ca 2ϩ affinity of the thin filament in a variety of ways; 1) directly (locally targeting the cTnC Ca 2ϩ affinity at the level of cTnC or proteins in direct contact) or 2) indirectly but through local interactions (involving proteins that contact cTnC through its interacting partners e.g. cTnI or cTnT) or by occurrences that indirectly influence cTnC Ca 2ϩ affinity such as strong cross-bridge formation (39,40).
The cTnC-A31S mutant increased the Ca 2ϩ affinity (⌬pCa 50 ϭ ϩ0.17) of the regulatory binding site II of isolated cTnC. In comparison, the previously characterized C84Y mutation also found in the N terminus did not measurably alter Ca 2ϩ affinity (⌬pCa 50 ϭ Ϫ0.01) of site II in isolated cTnC. The effects of the L29Q mutation on Ca 2ϩ affinity of site II indicate that cTnC-L29Q was less sensitive to Ca 2ϩ (⌬pCa 50 ϭ Ϫ0.12) than the cTnC-A31S mutant reported here (29). In addition, the C-terminal mutant cTnC-D145E increased Ca 2ϩ affinity of site II (⌬pCa 50 ϭ ϩ0.11) in a more complex manner, as it indirectly influences Ca 2ϩ binding. The other HCM-linked cTnC mutants N-terminal A8V and C-terminal E134D did not statistically change Ca 2ϩ affinity of site II in isolated cTnC (33).
Furthermore, we found that the cTnC-A31S mutant sensitized the thin filament to Ca 2ϩ (⌬pCa 50 ϭ ϩ0.54) to a much larger degree than any cTnC HCM mutants tested to date. In comparison, the previously described HCM cTnC mutants A8V and D145E increased thin filament Ca 2ϩ affinity with (⌬pCa 50 s of ϩ0.14 and ϩ0.08), respectively (33). It is well known that the number of cross-bridges can modulate both Ca 2ϩ sensitivity and TnC affinity for the thin filament (41)(42)(43)(44)(45)(46)(47). Therefore, the addition of myosin S1 was used to assess whether strong cross-bridge formation indirectly (ϪATP) modulated the Ca 2ϩ affinity of the mutant containing thin and thick filament differently than the WT (41). S1 decreased the ⌬pCa 50 of the cTnC-A31S thin filament compared with when it was in a lower level of complexity (thin filament alone), which indicates that the thin filament may not always be the best predictive model. Subsequently, Ca 2ϩ affinity of troponin with the addition of the thin filament and the catalytic myosin S1 more closely recapitulates what is seen in skinned fibers.
When combined, these data indicate that the A31S mutation increased Ca 2ϩ affinity of cTnC of this increasingly complex system through a direct mechanism, such as increased affinity of the cTnC regulatory domain for Ca 2ϩ or another known mechanism such as increased affinity of the mutant for cTnI. Potentially, a dramatically increased Ca 2ϩ affinity of cTnC has a higher likelihood of perturbing its Ca 2ϩ buffering role. The A31S mutant appears to substantially affect interactions  SEPTEMBER 14, 2012 • VOLUME 287 • NUMBER 38 between thin filament proteins, and this level may serve as the primary source of increased Ca 2ϩ sensitivity. In summary, the profoundly altered Ca 2ϩ binding properties of the cTnC-A31S mutant in the isolated state, cTn complex and thin filament coupled with early presentation of disease in the proband has never been shown before with a HCM-associated cTnC mutant.

TNNC1-A31S in HCM and Ventricular Fibrillation
Structural Characterization of the cTnC-A31S Mutant-Visualization of the troponin complex using the PyMol program suggested that A31S mutation may exert its Ca 2ϩ -sensitizing effects by locally stabilizing the EF-hand structure of the inactive Ca 2ϩ binding site I located in the N terminus (Fig. 6). The substitution of serine for alanine at position 31 introduces a polar amino acid into the Ca 2ϩ binding loop. Using a mutagenic function of the program without energy minimalization, substitution of Ser-31 in the mutant cTnC introduces a reactive hydroxyl group in close proximity to the backbone NH 2 group of Asp-33 (see Fig. 6A). The program predicts formation of an additional H-bond that would provide three H-bonds to stabilize the structure of the inactive Ca 2ϩ binding site (see Fig. 6B). It has been shown in skeletal troponin C that binding of Ca 2ϩ to the EF hands is cooperative, where coordination exists between the Ca 2ϩ binding sites of each domain. This same coordination has not been shown in the cTnC N terminus as Ca 2ϩ cannot bind to Site I. Therefore, we speculate that this mutation helps to order the structure of site I in a manner similar to Ca 2ϩ binding to site I in skeletal troponin C. This will need to be verified by established structural techniques that provide coordinates for the mutant protein compared with WT.
The CD data in the unbound (apo) state indicate that the mutation does not globally affect cTnC structure. No significant alterations were seen in the Mg 2ϩ -bound cTnC structure, indicating that the N-terminal mutation does not perturb the structure of the C terminus, as Mg 2ϩ binds primarily to the C terminus. The mutation also did not detectably alter the secondary structure of Ca 2ϩ /Mg 2ϩ -bound cTnC; this is not surprising as most of the spectral changes that occur when cTnC binds Ca 2ϩ originate from the C terminus (48,49). Therefore, we suggest that A31S in isolated cTnC sensitizes Ca 2ϩ binding to site II by locally affecting coordination between the Ca 2ϩ binding sites, which subtly affects overall structure, and results in a dramatic change in function.
In conclusion, the discovery of the TNNC1 mutation A31S represents one of the first cTnC mutants associated with verified episodes of ventricular fibrillation and aborted sudden cardiac death. This mutation may alter Ca 2ϩ handling and result in both hypertrophic and electrophysiologic remodeling of the cardiomyocyte. From our characterization, it appears that the A31S mutation may increase the Ca 2ϩ binding affinity of the regulatory site II of cTnC by stabilizing the N-domain structure. This in turn could bestow a tremendous capacity for Ca 2ϩ sensitization of the mutant containing myofilament. This mechanistic analysis coupled with clinical data provides insight on the source of myofilament dysfunction and the pathogenic nature of the mutation. It is evident that additional TNNC1 mutations will be discovered in the future and that this study along with others is essential to establish defined functional profiles characteristic of disease-causing mutations located within TNNC1.