Enhanced troponin I binding explains the functional changes produced by the hypertrophic cardiomyopathy mutation A8V of cardiac troponin C

Higher affinity for TnI explains how troponin C (TnC) carrying a causative hypertrophic cardiomyopathy mutation, TnC A8V , sensitizes muscle cells to Ca 2+ . Muscle fibers reconstituted with TnC A8V require ~2.3-fold less [Ca 2+ ] to achieve 50% maximum-tension compared to fibers reconstituted with wild-type TnC (TnC WT ). Binding measurements rule out a significant change in N-terminus Ca 2+ -affinity of isolated TnC A8V , and TnC A8V binds the switch-peptide of troponin-I (TnI sp ) ~1.6-fold more strongly than TnC WT ; thus we model the TnC-TnI sp interaction as competing with the TnI-actin interaction. Tension data are well-fit by a model constrained to conditions in which the affinity of TnC A8V for TnI sp is 1.5-1.7-fold higher than that of TnC WT at all [Ca 2+ ]. Mean ATPase rates of reconstituted cardiac myofibrils is greater for TnC A8V than TnC WT at all [Ca 2+ ], with statistically significant differences in the means at higher [Ca 2+ ]. To probe TnC-TnI interaction in low Ca 2+ , displacement of bis-ANS from TnI was monitored as a function of TnC. Whereas Ca 2+ -TnC WT displaces significantly more bis-ANS than Mg 2+ -TnC WT , Ca 2+ -TnC A8V displaces probe equivalently to Mg 2+ -TnC A8V and Ca 2+ -TnC WT , consistent with stronger Ca 2+ -independent TnC A8V -TnI sp . A Matlab program for computing theoretical activation is reported. Our work suggests that contractility is constantly above normal in hearts made hypertrophic by TnC A8V .


INTRODUCTION
Hypertrophic cardiomyopathy (HCM) is a disease of the myocardium in which the left ventricle wall becomes abnormally thick and stiff. Mutations in sarcomeric proteins have been reported as a primary source of dysfunction leading to the development of HCM in humans (1)(2)(3)(4)(5). Although sarcomeric protein mutations are considered "poison peptides" due to their negative impact on muscle regulation, little is known about the molecular mechanisms underlying changes in protein function. Within the sarcomere, TNNC1 (gene encoding cardiac troponin C, TnC) is a target for mutations associated with HCM (6)(7)(8)(9)(10). Of the missense mutations of TNNC1, A8V, causes the largest increase in Ca 2+ sensitivity of force development (7).
Cardiac troponin C function integrates with the exchange of Ca 2+ within the cytosol. Aside from buffering Ca 2+ in myocytes (11), TnC controls the kinetics of cardiac muscle activation and relaxation in response to association and dissociation of Ca 2+ (12). As a component of myofibrils, TnC participates with the transport mechanisms of the sarcoplasmic reticulum and sarcolemma to regulate and respond to transient Ca 2+ concentrations in the range of 10 −7 to 10 −5 M during rest and peak activation, respectively (13).
A divalent cation binding site (site II) in the N-terminal domain of TnC is specific for Ca 2+ (14), which allows the N-terminal domain to exist in predominantly a ligand-free or ligand-bound form during resting or activating states, respectively (15). Ligand binding to site II is energetically coupled to an equilibrium of closed and open conformations of the Nterminal domain (16). Ca 2+ binding destabilizes the secondary structure required for the closed conformation, thereby increasing the probability of the open conformation (17). The open conformation exposes a hydrophobic patch that interacts with H3(I) of cardiac troponin I (TnI) (Fig. 1). H3(I) together with H4(I) comprise a larger switch peptide (TnI sp ) (16,18) that, along with the C-terminal domain of TnI, stabilizes troponin (Tn) and tropomyosin (Tm) in the position that blocks activation during rest (19)(20)(21). To activate contraction, TnI sp rotates away from actin to bind the open conformation of TnC (18). TnI sp binds the hydrophobic patch during the time that the N-terminal domain of TnC is in the open state, by a conformational selection mechanism (22)(23)(24). Baseline and maximum activation reflect the distributions of closed and open conformations, which have probabilities that depend on Ca 2+ binding.
Although force measurements suggest that the A8V mutation acts by increasing the Ca 2+ binding to N-terminal of domain of TnC as the primary cause of the disease (7), structural and functional measurements do not support this mechanism. No significant difference in Nterminal domain Ca 2+ affinity is observed between isolated TnC A8V and wild-type (WT) TnC (TnC WT ) (25). Instead, the A8V mutation shifts the conformation of the N-terminal domain in favor of the open conformations of ligand-bound and ligand-free TnC (26). The A8V mutation is located in the N-helix of the crystal structure of TnC WT and is situated between site II for Ca 2+ and the H3(I) interaction site ( Fig. 1) (18). A synthetic peptide corresponding to TnI SP binds to TnC A8V ~1.6-fold more strongly than to TnC WT (27). Based on these observations, we hypothesize that altered interactions between TnC and TnI SP is the primary source of the changes in function observed for myofilaments carrying TnC A8V (e.g., increase myofilament Ca 2+ sensitivity). We present a model to reconcile this hypothesis with the paradoxical increased sensitivity to Ca 2+ observed in force measurements (7).

Three-dimensional Visualization
The location of the TnC A8V mutation within the 52-kDa Tn complex was visualized in the 1J1E Protein Data Bank file using PyMol software. PyMol is an open source molecular visualization program that allows manipulation of PDB files containing molecular coordinates obtained from structures based on x-ray crystallography or nuclear magnetic resonance.

Protein extraction and reconstitution
Native TnC was depleted from myofibrils using a CDTA extraction buffer (5 mM CDTA, 5 mM DTT, pH 8.4) for ~2.5 h at room temperature. After each 30 min of incubation, the myofibrils were centrifuged (5 min at 750 × g and 4°C), the supernatant was discarded, and a new CDTA extraction solution was added. To remove CDTA, myofibrils were centrifuged (5 min at 1,500 × g and 4°C) and resuspended in wash buffer three times. Native TnCextracted myofibrils were reconstituted with TnC WT or TnC A8V (1.0 mg/ml) for 1 h at 4°C. Non-specifically bound TnC was removed from the reconstituted samples by several washes (centrifugation and resuspension) using wash buffer. Myofibril concentrations were determined using a Pierce TM Coomassie Plus (Bradford) assay kit.

Myofibrillar ATPase activity
The ATPase assay was performed at various pCa values using solutions containing 2 mM EGTA, 3 mM NTA, 20 mM MOPS, 1 mM free Mg 2+ , ~2.5 mM MgATP 2− , pH 7.0 with ionic strength fixed to 80 mM (adjusted with KCl) and selected Ca 2+ concentrations. The free Ca 2+ in the ATPase solutions were calculated using pCa calculator software (30). The pCa values of the solutions were 8.0, 7.0, 6.6, 5.8, and 5.0 (1.0 × 10 −8 M, 1.0 × 10 −7 M, 2.51 × 10 −7 M, 1.58 × 10 −6 M and 1.0 × 10 −5 M free Ca +2 , respectively). The myofibrillar concentration was 0.4 mg/ml, and the experiments were carried out at 25°C. The reaction was initiated by adding ATP and quenched after 7 min with the addition of ~4.6% trichloroacetic acid. The precipitated proteins were collected by centrifugation (7 min at 3,095 × g and 4°C). Inorganic phosphate released by ATP hydrolysis was measured in the supernatant using the colorimetric method of Fiske and Subbarow (31).

Fluorescence assay
TnC WT , TnC A8V , and TnI were dialyzed into fluorescence buffer containing 120 mM MOPS, 100 mM KCl, and 2 mM EGTA at pH 7.0. For experiments in the presence of Mg 2+ and Ca 2+ , MgCl 2 and CaCl 2 were added before titration to yield final concentrations of 2 mM free Mg 2+ and 0.1 mM free Ca 2+ . For experiments in the presence of Mg 2+ only, MgCl 2 was added to a final concentration of 2 mM free Mg 2+ . In addition, before titration, freshly prepared 1 mM DTT was added to the buffer. TnI and 4,4′-dianilino-1,1′-binaphthyl-5,5′disulfonic acid, dipotassium salt (bis-ANS) were fixed at 0.5 mM. TnC WT or TnC A8V was titrated at every 0.05 μM until reaching 0.5 μM, followed by titration of every 0.1 μM until the final concentration of 1 μM was reached. TnC and TnI concentrations were determined by spectroscopy at 280 nm (NanoDrop ND-1000 spectrophotometer) with extension coefficients of 4,595 and 9,770 cm −1 M −1 , respectively. The steady-state fluorescence measurements were performed in a Jasco FP-8300 spectrofluorometer at a constant photomultiplier (PMT) voltage of 340 V. The fluorescent probe bis-ANS was excited at 390 nm, emission was detected in a wavelength range of 450-520 nm, and the highest peak intensity was recorded. The experiments were carried out at 25°C and under constant stirring (600 rpm). A baseline experiment was performed in the absence of TnI to measure the fluorescence changes upon binding of TnC to bis-ANS. At the PMT voltage of 340V, minimum changes in fluorescence emission (A.U.) were observed in the baseline titration. The data obtained were then subtracted from the baseline. Data were analyzed and fit using linear regression.

Statistical analyses
The data were analyzed for significance using Student's t-test (unpaired). The results were considered statistically different when p < 0.05. The data are expressed as means ± S.E.

Modeling
We model regulation as modulation of Tm in the activated state (M) by multiple equilibria (32). In this model, M forms in the activated position of the thin filament as a ternary complex of myosin, Tm, and actin (33,34) from Tm in the central position of the thin filament (C) by a spontaneous process (equilibrium pathway). The equilibrium is perturbed by the crossbridge cycle in the physiologic muscle by an additional kinetic pathway. The kinetic pathway operates continuously with fixed non-zero probability to regenerate M without sampling C. Failure to regenerate returns Tm to the central position. The kinetic pathway is responsible for establishing an ensemble of Tm molecules that function as a unit in the manner predicted by theory (32) and consistent with the observation that an initial binding of two myosin heads establishes a regulatory unit that can accommodate binding by a much larger number of secondarily bound myosin heads (35). The combination of equilibrium and kinetic pathways are expressed in the model as ( Fig. 2), where K 0 ′ includes the number of Tm molecules functioning as an ensemble (n > 1). In addition, the odds of regenerating M without decay to C (α) is included in the mathematical expression of M, (1) that is derived for the steady-state (36). C is the only Ca 2+ -dependent variable of Eq. 1 with K 0 ′ , α, and n each having fixed values determined empirically from WT data (see Table 1).
Regulation is achieved by controlling the availability of C for M formation (Eq. 1). C is By detailed balance (Fig. 2), the following relationship must hold,

Analysis of tension data
Fibers reconstituted with TnC A8V require less Ca 2+ to achieve a given fraction of maximum tension than fibers reconstituted with TnC WT (7). Because site II of TnC WT and TnC A8V have indistinguishable affinities for Ca 2+ (25), enhanced sensitivity to Ca 2+ is more likely explained by strengthened interaction between TnC and TnI sp . We model no change in Ca 2+ affinity by holding K 2 and K 4 constant and an increase in TnI sp affinity as a reduction in the apparent affinity of TnI sp for actin, i.e. reduction in K 1 and K 3 (Fig. 2). Holding K 2 /K 4 constant implies that K 1 /K 3 is constant (Eq. 2).
To vary only K 1 and keeping K 1 /K 3 constant, values for the other parameters must be fixed in Eq. 1. For simplicity, K 0 ′ is set to unity and n, and α have values used previously ((32); see also Table 1). Because K 2 governs the binding of Ca 2+ to uncoupled Tn (Fig. 2), K 2 is set approximately equal to the measured affinity constant for isolated Tn, and, hence, K 4 is given by K 2 K 3 /K 1 (Eq. 2). We compare K 2 and K 1 /K 3 values derived previously for skeletal muscle (32) with substantially greater values of each as a means of testing sensitivity (Table  1). For both lower and upper boundary conditions, parameter K 1 is adjusted to achieve theoretical activations that fit Ca 2+ -dependent tension data normalized between 0% and 100% maximum (Fig. 3A, B). A 1.5-to 1.7-fold decrease in the bench-mark K 1 shifts the theoretical Ca 2+ activation curve to align with the tension data collected for fibers reconstituted with TnC A8V (Fig. 3A, B). By Eq. 2 and the constraint imposed on K 2 and K 4 , the model predicts that K 3 also must decrease 1.5-to 1.7-fold. Although a unique solution is not established, this analysis reconciles the shift observed in Ca 2+ sensitive force (7) and the absence of measured change in Ca 2+ binding affinity in isolated TnC (25). A Matlab program to reproduce and extend the modeling shown here is included in Supplementary  Material (Fig. S1). As a control, we fit a 2-fold increase in Ca 2+ activation data published for the L48Q mutation of TnC by holding K 1 constant and increasing K 2 , consistent with the increase in affinity of Tn reported for this mutation (Supplementary Material, Fig. S2).
On an absolute scale, the predicted activation is greater for all Ca 2+ concentration between baseline and maximum fractional activations of 0.1-0.9 and 0.05-0.95 for lower and upper boundary conditions, respectively (Fig 3C, D concentrations causes the leftward shift in the relative force curve (Fig. 3A, B).

Activation of myofibrillar ATPase
To test the model's prediction on an absolute scale, we measured Ca 2+ -dependent ATPase rate of cardiac myofibrils reconstituted with TnC A8V and TnC WT . Given midrange Ca 2+ concentration, i.e., pCa 5.8, ATPase rates of myofibrils reconstituted with TnC WT and TnC A8V are 48.7% and 62.5% of maximum, respectively (Table 2). This difference is consistent with the enhancement observed in the tension record (Fig. 3A). Given various Ca 2+ concentrations, all conditions stimulated higher mean ATPase rates for myofibrils reconstituted with TnC A8V than myofibrils reconstituted with TnC WT (Fig. 4). Differences in the means are statistically significant for Ca 2+ concentrations above the midrange (Table  2). Elevation in ATPase rate at the extremes of Ca 2+ concentration is consistent with a general increase in contractility brought about by the interaction between the more open TnC A8V and TnI conformations.

Titration of fluorescence associated with TnI
To detect subtle differences in the interaction between TnC and TnI, we monitored the fluorescence of bis-ANS. Compared with bis-ANS bound to TnI, unbound bis-ANS has much reduced fluorescence intensity. We interpret reduction in fluorescence intensity as owing to the displacement of bis-ANS that results from complex formation between TnC and TnI. Rather than a typical hyperbolic binding curve, we observe a linear decrease in fluorescence as a function of added TnC (Fig. 5). Specific binding of TnC is evident from the saturation of the fluorescence decrease for additions that exceed 1 mol TnC/mol TnI (Fig. 5 insets). The appearance of irreversible binding may be explained by a combination6 of high affinity mass action binding (37) and persistent weak interactions that slow the diffusive separation of unbound proteins. To distinguish between the functions of the domains of TnC, we conducted the assay in two Ca 2+ concentrations. Letting Mg-TnC WT and Ca-TnC WT represent the structures of TnC in low or saturating Ca 2+ concentrations (see introduction), both structures are expected to form high-affinity interactions between the Cterminal domain of TnC and TnI. However, only Ca-TnC WT is expected to promote a secondary interaction between the N-terminal domain of TnC and TnI. As expected, the more extensively bound Ca-TnC WT displaces proportionately more bis-ANS than Mg-TnC WT (Fig. 5A). By contrast, Ca-TnC A8V and Mg-TnC A8V displace nearly the same amount of probe (Fig. 5B) and have nearly the same slope as Ca-TnC WT . Thus, compared with Mg-TnC WT , the N-terminal domain of Mg-TnC A8V appears to have a more stable association with TnI, consistent with having a more open N-terminal domain conformation independent of Ca 2+ -binding at site II.

DISCUSSIONS
We find that a modest increase in affinity between the N-terminal domain of TnC and the switch region of TnI causes the observed shift in midrange tension output to lower Ca 2+ concentration observed in cardiac muscle containing TnC A8V . Evidence in support of this hypothesis comes from the combination of experimental and modeling approaches. Measurement of binding rules out a significant change in Ca 2+ binding affinity, and structural modeling rules out mutagenesis directed to the site of interaction between TnC and TnI. Based on a kinetic model of muscle regulation, we determined that the observed enhancement in Ca 2+ -sensitivity is explained by 1.5-to 1.7-fold greater affinity between TnC and TnI SP , which matches the experimental measurement of 1.6 fold (27). Average myofibril ATPase rates are higher for myofibrils reconstituted with TnC A8V at all Ca 2+ concentrations tested, and are significantly higher for Ca 2+ concentrations above the titration midpoint. Compared with TnC WT , TnC A8V displaced more fluorescent probe from TnI in the absence of saturating Ca 2+ , consistent with a stronger interaction.
Our model is distinct in the manner by which Ca 2+ participates in attaining the activated state. Standard models derived from Hill et al. (38) are fundamentally allosteric and hence propose structural changes in Tm owing to a series of structural changes initiated by Ca 2+ binding to site II of TnC. Accordingly, the states of Tm corresponding to the B, C, and M positions result from Ca 2+ -free and myosin unbound; Ca 2+ -bound and myosin weakly bound to actin; and, Ca 2+ -bound and myosin strongly bound to actin, respectively (39)(40)(41).  (Fig. 2).
Whereas the activated states of allosteric models require contribution of binding by both Ca 2+ and strong-binding crossbridges, the activated state of our model requires no direct participation of Ca 2+ binding. Instead, the activated state requires only Tm in the C position and force-bearing cycling crossbridges, consistent with consensus observations (41). Cooperative activation (39) without direct participation of ligand, i.e., non-allosteric cooperativity, is a hallmark of a second chance mechanism (36). The statistical basis of a second chance mechanism can be linked to fundamental thermodynamic properties of systems that couple equilibrium and kinetic forces, such as forces acting on Tm by cycling crossbridges (42). Absent experimental proof of a Ca 2+ -driven conformation of Tm being required for the activated state of the physiologic muscle, the simplest explanation is that Ca 2+ binding is required only to perturb the equilibrium of closed and open states of TnC.
To date, three distinct probands carrying the Ala8 to Val mutation in TnC have been reported ((7,43) and personal communication). Although one of the probands reported a parent who was also positively genotyped for the mutation, the available genetic data remain difficult to interpret. In one clinical report, the mutation arose de novo; in another report, the proband carried two mutations in different sarcomeric proteins (7,43). The functional characterization is crucial to ascertain whether the A8V mutation in TnC is a true pathogenic variant and how it exerts its negative effect on muscle regulation.
Functional assays conducted with reconstituted proteins have shown that when the A8V mutant TnC is incorporated in the thin filament, it increases Ca 2+ sensitivity of the myofilament. Kinetic measurements in reconstituted thin filament have suggested that a slower Ca 2+ off rate might be the cause of the increased Ca 2+ binding affinity to the Nterminal domain of TnC A8V (28,44). Activation and deactivation of muscle is controlled by Ca 2+ binding and dissociating from the TnC N-terminal domain, which subsequently interacts with the C-terminal domain of TnI. Therefore, not only Ca 2+ kinetics but also TnC and TnI affinity are crucial in the regulation of cardiac muscle contraction. Little is known about how TnC A8V delays the Ca 2+ off rate and whether this involves an abnormal interaction between TnC and TnI. One simplistic explanation supported by our model is that the N-terminal domain of TnC A8V binds with greater affinity to the TnI C-terminal domain. The fluorescence binding studies reported here are in agreement with a previous report (27) and support the hypothesis that TnC A8V and TnI bind to each other at a higher affinity during resting conditions, when the muscle should be fully relaxed.
The consequences of increased binding affinity between TnC A8V and TnI may have broader physiological implications. The functional consequences of the A8V substitution in TnC were evaluated in a knock-in (KI) mouse (45). Intact cardiomyocytes isolated from both heterozygous and homozygous KI mice had slower sarcomere length relaxation time and Ca 2+ decay at different frequencies of stimulation. Furthermore, homozygous mice showed signs of diastolic dysfunction and a hypercontractile phenotype, e.g., increased ejection fraction (45). The in vivo data can be correlated with the myofibrillar ATPase findings since higher myofibrillar ATPase activation can indicate a greater degree of muscle activation. Increased actomyosin ATPase activation was also observed in an early report (44). Here, we observed increased mean myofibrillar ATPase activity at all low Ca 2+ concentrations tested, although statistical significance could not be demonstrated for all Ca 2+ concentrations, reflecting sensitivity limits of the assay. We interpret an effect of the TnC-TnI interaction on myofibrillar ATPase as contributing to the contractility of cardiac myocytes. Hence, even a small but persistent increase in myofibrillar ATPase at low Ca 2+ levels may be sufficient to cause hypertrophy of the muscle over time.
In conclusion, these results suggest that the effect of the A8V mutation in delaying the Ca 2+ off rate from TnC would be secondary to altering the interaction between TnC and TnI. This is an important observation because new therapies could be tailored to specifically target binding between TnC and TnI, rather than intracellular Ca 2+ concentration or Ca 2+ binding to TnC. The results obtained in this work do not allow us to determine in exact nature of the altered interactions between TnC and TnI; however, if the defect lies in the kinetics of binding to TnI rather than dissociation from TnI, this could be an important site for the development of a therapeutic target.

3)
Theoretical predictions of enhanced TnI-TnC interaction recapitulate experimental published data

5)
Permanent increase in contractility is the presumptive cause of hypertrophic cardiomyopathy    Data from chemically skinned and TnC-depleted cardiac fibers are reproduced from an earlier study (7)