Rat cardiac troponin T mutation (F72L)-mediated impact on thin filament cooperativity is divergently modulated by α- and β-myosin heavy chain isoforms

The emergence of divergent thin filament cooperativity, induced by α- and β-myosin heavy chain (MHC), engenders a mutant cardiac troponin T (TnT_F70L) to produce contrasting effects on contractile dynamics. TnT_F70L causes a dilated cardiomyopathy-like phenotype in the presence of α-MHC and a hypertrophic cardiomyopathy-like phenotype in the presence of β-MHC.

Abstract

The primary causal link between disparate effects of human hypertrophic cardiomyopathy (HCM)-related mutations in troponin T (TnT) and α- and β-myosin heavy chain (MHC) isoforms on cardiac contractile phenotype remains poorly understood. Given the divergent impact of α- and β-MHC on the NH₂-terminal extension (44–73 residues) of TnT, we tested if the effects of the HCM-linked mutation (TnT_F70L) were differentially altered by α- and β-MHC. We hypothesized that the emergence of divergent thin filament cooperativity would lead to contrasting effects of TnT_F70L on contractile function in the presence of α- and β-MHC. The rat TnT analog of the human F70L mutation (TnT_F72L) or the wild-type rat TnT (TnT_WT) was reconstituted into demembranated muscle fibers from normal (α-MHC) and propylthiouracil-treated (β-MHC) rat hearts to measure steady-state and dynamic contractile function. TnT_F72L-mediated effects on tension, myofilament Ca²⁺ sensitivity, myofilament cooperativity, rate constants of cross-bridge (XB) recruitment dynamics, and force redevelopment were divergently modulated by α- and β-MHC. TnT_F72L increased the rate of XB distortion dynamics by 49% in α-MHC fibers but had no effect in β-MHC fibers; these observations suggest that TnT_F72L augmented XB detachment kinetics in α-MHC, but not β-MHC, fibers. TnT_F72L increased the negative impact of strained XBs on the force-bearing XBs by 39% in α-MHC fibers but had no effect in β-MHC fibers. Therefore, TnT_F72L leads to contractile changes that are linked to dilated cardiomyopathy in the presence of α-MHC. On the other hand, TnT_F72L leads to contractile changes that are linked to HCM in the presence of β-MHC.

NEW & NOTEWORTHY

The emergence of divergent thin filament cooperativity, induced by α- and β-myosin heavy chain (MHC), engenders a mutant cardiac troponin T (TnT_F70L) to produce contrasting effects on contractile dynamics. TnT_F70L causes a dilated cardiomyopathy-like phenotype in the presence of α-MHC and a hypertrophic cardiomyopathy-like phenotype in the presence of β-MHC.

healthy heart function depends on the magnitude and dynamics (e.g., speed) of myofilament activation, because they strongly depend on cooperative mechanisms operating within the myofilament system (5, 6, 41). Cardiomyopathy mutations in contractile proteins may affect cooperative mechanisms mediated by protein-protein interactions, leading to abnormal heart function. Previous studies have shown that cardiac troponin (Tn) T (TnT) is an essential thin filament regulatory protein required for full expression of thin filament cooperativity (23, 30, 45, 49). We recently demonstrated that a specific domain, the NH₂-terminal extension (NTE; 43–71 residues), is important for human TnT to exert its full effect on thin filament cooperativity (21, 32). Three different mutations, D46V (28), K66Q (39), and F70L (42), which are linked to familial hypertrophic cardiomyopathy (HCM), have been mapped to the NTE of human TnT. However, the functional characterization of these HCM-linked mutations is lacking.

The main focus of this study is to characterize the effects of the human HCM mutation F70L, which is located in the NTE of human TnT. Our recent study indicated that the NTE plays an important role in cooperative activation of cardiac thin filaments (21, 32). We suggested that the NTE exerts its influence on activation via its impact on the tropomyosin (Tm)-Tn complex, which forms one regulatory unit (RU) on the thin filament. Because hydrophobic amino acids determine the packing and structure of a protein, any hydrophobicity-related changes (e.g., F70L) may alter the packing of the NTE to modulate the intramolecular interactions of the extreme NH₂ terminus (upstream 1–42 residues) with the central region (CR; 77–193 residues) of TnT (20) and/or CR (downstream)-Tm activity. Because the CR-Tm interaction is essential for thin filament activation, such altered intra- and/or intermolecular interactions may in turn modify the CR-Tm activity to tune the magnitude and/or dynamics of thin filament activation. Given the importance of CR-Tm activity in tuning thin filament activation and how such actions are differentially altered by α- and β-myosin heavy chain (MHC) isoforms (18, 22, 31, 35, 50), we hypothesized that the effects of human TnT_F70L would be differentially altered by α- and β-MHC.

Differential impact of α- and β-MHC on thin filament cooperativity stems mainly from their different kinetic properties (16, 44, 46). Differences in kinetic properties of the faster-cycling α-MHC and the slower-cycling β-MHC lead to disparate effects on thin filament cooperativity, because the dwell time of strongly bound cross bridges (XBs) varies with kinetic rates. The magnitude of the effect of the fast-cycling α-MHC on thin filament cooperativity is expected to be lower than that of the slow-cycling β-MHC, because α-MHC remains bound to the thin filament for a shorter period than does β-MHC. Because full activation of the thin filament is dependent on the Tn- and XB-mediated effects, the interplay between the TnT- and MHC isoform-induced changes in thin filament cooperativity will have a significant impact on the magnitude and dynamics of myofilament activation. A logical extension of this argument strongly suggests that the TnT_F72L-induced effect in the thin filament is differently modulated by α- and β-MHC, leading to divergent effects on cardiac myofilament function.

To test our hypothesis, we generated a recombinant rat TnT analog (TnT_F72L) of the human F70L mutation. TnT_F72L and wild-type rat TnT (TnT_WT) were reconstituted into detergent-skinned left ventricular papillary muscle fibers extracted from normal rat hearts expressing α-MHC and propylthiouracil (PTU)-treated rat hearts expressing β-MHC. Steady-state and dynamic contractile parameters were measured in α- and β-MHC fibers that were reconstituted with Tn containing TnT_WT or TnT_F72L. TnT_F72L had contrasting effects on Ca²⁺-activated maximal tension and myofilament Ca²⁺ sensitivity [−log of free Ca²⁺ concentration (pCa) required for half-maximal activation (pCa₅₀)] in α- and β-MHC fibers; for instance, tension and pCa₅₀ were attenuated in α-MHC fibers but increased in β-MHC fibers. TnT_F72L had contrasting effects on the rate constants of XB recruitment dynamics (b) and force redevelopment (k_tr) in α- and β-MHC fibers. TnT_F72L increased the rate of XB detachment kinetics in α-MHC fibers but had no effect in β-MHC fibers. Novel findings from our study suggest that TnT_F72L induces contrasting functional phenotypes in the presence of α- and β-MHC. In this study we discuss the molecular mechanisms by which TnT_F72L leads to divergent functional outcomes in the presence of α- and β-MHC.

METHODS

Animal Treatment Protocols

The treatment of animals used in this study followed the established guidelines approved by the Washington State University Institutional Animal Care and Use Committee. Left ventricular papillary muscle bundles were isolated from 3- to 4-mo-old normal (α-MHC) and PTU-treated (β-MHC) male Sprague-Dawley rats. Rats were carefully handled to minimize pain and suffering, in accordance with the established guidelines of the National Academy of Sciences Guide for the Care and Use of Laboratory Animals.

PTU Treatment

We administered PTU in both drinking water (0.2 g/l) and solid feed (0.15% PTU; Harlan Laboratories, Madison, WI) for ∼5 wk. PTU-induced hypothyroidism has been shown to silence the α-MHC promoter, leading to activation of the β-MHC promoter in the cardiac ventricle (12). This results in the complete switch of MHC isoform from α- to β-MHC protein in the ventricle.

Detergent-Skinned Cardiac Muscle Fibers

Detergent-skinned cardiac muscle fibers were isolated as described elsewhere (9, 10). Hearts from rats deeply anesthetized with isoflurane were quickly removed and placed into an ice-cold high relaxing (HR) solution containing (in mM) 20 2,3-butanedione monoxime, 50 N,N-bis (2-hydroxyethyl)-2-aminoethane sulfonic acid (BES), 20 EGTA, 6.29 MgCl₂, 6.09 Na₂ATP, 30.83 potassium propionate, 10 sodium azide, 1.0 DTT, and 4 benzamidine-HCl. The pH of the HR solution was adjusted to 7.0 with KOH. HR solution also included protease inhibitors (in μM: 5 bestatin, 2 E-64, 10 leupeptin, 1 pepstatin, and 200 PMSF). Left ventricular papillary muscle bundles were dissected into smaller (∼0.15 mm wide and 2 mm long) muscle fiber bundles in ice-cold solution. Muscle fibers were chemically skinned overnight at 4°C in HR solution containing 1% Triton X-100.

MHC Composition and the Phosphorylation Status of Sarcomeric Proteins in Normal and PTU-Treated Rat Hearts

The left ventricular tissue was rapidly frozen in liquid nitrogen, thoroughly crushed, and minced using a pestle and mortar (18). One milligram of minced tissue was solubilized in 10 μl of protein extraction buffer [2.5% SDS, 10% glycerol, 50 mM Tris base (pH 6.8 at 4°C), 1 mM DTT, 4 mM benzamidine-HCl, and protease inhibitors (E-64, leupeptin, and bestatin)]. Solubilized tissue was homogenized on ice using a Tissue Tearor (model 985370-395, Biospec Products), sonicated in a water bath at 4°C, and centrifuged at 10,000 rpm (9,300 g). Normal and PTU-treated cardiac muscle samples were electrophoresed on a large 4% SDS gel to determine the MHC isoform composition (9, 16, 22). For assessment of the phosphorylation status of sarcomeric proteins, equal quantities of normal and PTU-treated rat heart samples were loaded and run on a small 12.5% SDS gel. The gel was then treated with Pro-Q Diamond stain and destain (catalog nos. P33300 and P33310, Life Technologies, Grand Island, NY) according to the manufacturer's protocol. The gel was imaged using UV transillumination and a Bio-Rad ChemiDoc XRS camera, and the resulting protein profiles were used to assess the differences in the phosphorylation levels of various sarcomeric proteins between normal and PTU-treated rat hearts (18, 21).

Purification of Recombinant Rat Cardiac Tn Subunits

Recombinant TnT (TnT_WT and TnT_F72L), rat cardiac Tn I (TnI), and rat cardiac Tn C (TnC) were purified as described elsewhere (8, 24, 40). TnT_WT and TnT_F72L were tagged with the c-myc epitope at the NH₂ terminus (11, 22, 36, 48). The expression vector was pSBETa, and the expression cell was BL21 Star DE. Briefly, TnT_WT and TnT_F72L were purified by ion-exchange chromatography (8, 10) on a DEAE-Sepharose fast-flow column (GE Healthcare Biosciences, Pittsburgh, PA). TnI was purified by ion-exchange chromatography on a CM Sepharose column (19, 24, 32). TnC protein was purified by ion-exchange chromatography as described previously (19, 32, 40). Samples from eluted fractions were run on a 12.5% SDS gel to determine their purity. Pure fractions were pooled and dialyzed extensively against deionized water containing 15 mM β-mercaptoethanol, lyophilized, and stored at −80°C.

Reconstitution of Tn Into Detergent-Skinned Rat Cardiac Muscle Fibers

Tn was reconstituted into muscle fibers as described elsewhere (9, 22). We used c-myc-tagged TnT_WT and TnT_F72L so that incorporation of the exogenously added subunits could be assessed by the differential gel migration pattern of c-myc-tagged proteins. The c-myc epitope at the NH₂ terminus of TnT does not affect cardiac function (36, 48). TnT_WT or TnT_F72L (0.9 mg/ml, wt/vol) and TnI (1.0 mg/ml, wt/vol) were dissolved in buffer 1 [50 mM Tris·HCl (pH 8.0), 6 M urea, 1.0 M KCl, 10 mM DTT, and a cocktail of protease inhibitors]. High salt and urea were removed by successive dialysis against buffer 2 [50 mM Tris·HCl (pH 8.0), 4 M urea, 0.7 M KCl, 0.2 mM PMSF, 2 mM benzamidine-HCl, 1 mM DTT, and 0.01% NaN₃], buffer 3 [50 mM Tris·HCl (pH 8.0), 2 M urea, 0.5 M KCl, 0.2 mM PMSF, 2 mM benzamidine-HCl, 1 mM DTT, and 0.01% NaN₃], and, finally, an extraction buffer [buffer 4; 50 mM BES (pH 7.0 at 20°C), 200 mM KCl, 10 mM 2,3-butanedione monoxime, 6.27 mM MgCl₂, 5 mM EGTA, 0.2 mM PMSF, 2 mM benzamidine-HCl, 1 mM DTT, and 0.01% NaN₃]. A fresh cocktail of protease inhibitors was added to the supernatant after the final dialysis. The protein solution was spun at 3,000 rpm (1,962 g) for 30 min to remove any undissolved protein. Detergent-skinned fibers were treated with the extraction solution containing TnT_WT + TnI or TnT_F72L + TnI for ∼3 h at room temperature (20°C) with constant stirring. Muscle fibers were washed three times using buffer 4 for 10 min at room temperature. TnT_WT + TnI- or TnT_F72L + TnI-treated fibers were then incubated with TnC (3.0 mg/ml, wt/vol) overnight at 4°C.

Western Blotting

Muscle fibers were denatured and solubilized in 2% SDS solution (10 μl/fiber), and an equal volume of protein loading dye [125 mM Tris·HCl (pH 6.8), 20% glycerol, 2% SDS, 0.01% bromophenol blue, and 50 mM β-mercaptoethanol] was added. Proteins were separated on an 8% SDS gel and transferred onto a PVDF membrane for Western blot analysis using a Trans-Blot Turbo transfer system (Bio-Rad Laboratories, Hercules, CA). The incorporation of TnT_WT or TnT_F72L was assessed using a monoclonal anti-TnT primary antibody (catalog no. M401134, Fitzgerald Industries, Concord, MA) followed by a HRP-labeled anti-mouse secondary antibody (catalog no. RPN 2132, Amersham Biosciences, Piscataway, NJ). Densitometric analysis was performed using NIH ImageJ software (http://rsbweb.nih.gov/ij/).

Steady-State Isometric Tension and ATPase Activity

Isometric tension and ATPase activity were measured as described previously (7, 9, 14, 47). T-shaped aluminum clips were used to attach the muscle fiber between a motor arm (catalog no. 322C, Aurora Scientific, Aurora, ON, Canada) and a force transducer (AE 801, Sensor One Technologies, Sausalito, CA). The sarcomere length (SL) of the muscle fibers was set to 2.3 μm under relaxing conditions, as described previously (7, 9, 14, 47). After two cycles of maximal activation and relaxation, the SL was readjusted to 2.3 μm if necessary. The muscle fiber was exposed to various Ca²⁺ solutions in a constantly stirred chamber. The concentration of Ca²⁺ ranged from pCa 4.3 to 9.0. The composition of pCa solutions was calculated using methods described previously (15). The composition of the maximal Ca²⁺ activation solution (pCa 4.3) was (in mM) 31 potassium propionate, 5.95 Na₂ATP, 6.61 MgCl₂, 10 EGTA, 10.11 CaCl₂, 50 BES, 5 sodium azide, and 10 phosphoenolpyruvate (PEP). The relaxing solution (pCa 9.0) contained (in mM) 51.14 potassium propionate, 5.83 Na₂ATP, 6.87 MgCl₂, 10 EGTA, 0.024 CaCl₂, 50 BES, 5 NaN₃, and 10 PEP. The activation and relaxing solutions also contained 0.5 mg/ml pyruvate kinase (500 U/mg), 0.05 mg/ml lactate dehydrogenase (870 U/mg), 20 μM diadenosine pentaphosphate, 10 μM oligomycin, and a cocktail of protease inhibitors. The pH and ionic strength of maximal activation and relaxing solutions were adjusted to 7.0 and 180 mM, respectively. All measurements were made at 20°C.

Steady-state isometric ATPase activity was measured according to a protocol described previously (14, 21, 27, 43, 47). Briefly, near-UV (340-nm) light was projected through the muscle chamber and then split (50:50) via a beam splitter and detected at 340 nm (sensitive to changes in NADH) and 400 nm (insensitive to changes in NADH). ATPase activity was measured as follows: ATP regeneration from ADP was coupled to the breakdown of PEP to pyruvate and ATP catalyzed by pyruvate kinase, which was linked to the synthesis of lactate catalyzed by lactate dehydrogenase. The breakdown of NADH was proportional to the ATP consumption and was measured by changes in UV absorbance at 340 nm. The signal for NADH was calibrated by multiple injections of 250 pmol of ADP (14, 21, 27, 43, 47).

Muscle Fiber Mechanodynamics

Muscle fibers were maximally activated at pCa 4.3 and subjected to steplike length perturbations on the order of ±0.5%, ±1.0%, ±1.5%, and ±2.0% of the initial muscle length (ML), as described previously (17). A nonlinear recruitment distortion (NLRD) model was fit to the elicited force responses to estimate several important model parameters: the magnitude of the instantaneous muscle fiber stiffness caused by a sudden change in ML (E_D), the rate by which the strain within bound XBs dissipates following a sudden change in ML (c), the parameter that characterizes the XB strain-mediated negative impact on other force-bearing XBs (γ), and the rate at which new XBs are recruited into the force-bearing state due to a change in ML (b). The relationship between model parameters and physiological phenomena is described as follows.

E_D.

In phase 1, a sudden increase in ML (Fig. 1A) causes an instantaneous increase in force, from steady-state isometric force (F_ss) to force at the end of phase 1 (F₁; Fig. 1B). The instantaneous increase in force is due to 1) rapid distortion of the elastic elements in the strongly bound XBs and 2) a change in muscle length (ΔL) that is too fast to allow recruitment of new XBs. An increase in F₁ indicates an increase in the number of strongly bound XBs; conversely, attenuation of F₁ indicates a decrease in the number of strongly bound XBs. Therefore, E_D is an approximate index of the number of strongly bound XBs and is estimated as the slope of the (F₁ − F_ss)-ΔL relationship (17).

Fig. 1.

Force response to a large-amplitude [+2% of initial muscle length (ML)] steplike length perturbation in maximally activated rat cardiac muscle fiber. Muscle fiber mechanodynamic experiments were carried out as described previously (17). A: large-amplitude steplike length perturbation imposed on the muscle fiber. B: representative experimental record showing characteristic features of the corresponding force transient. Force data were normalized by the isometric steady-state value (F_ss) just prior to stretch. F₁, magnitude of the instantaneous force increase at the end of phase 1; c, dynamic rate by which the instantaneous force decays during phase 2; γ, minimum in the force decline (nadir) before it begins to rise slowly; b, dynamic rate at which force rises to a new steady state (F_nss) during phase 3. Physiological significance of F₁, c, γ, and b is described in methods.

c.

Phase 2 is the rapid decay in force to a minimum (nadir; Fig. 1B) as the fiber is held at the increased ML (Fig. 1A). Force decays rapidly, because strained XBs detach and equilibrate into a non-force-bearing state. The dynamic rate at which the force decays (c) has been demonstrated to be an index of the XB detachment rate constant (g) (4, 17).

γ.

The magnitude of force decline to a minimum in phase 2 (nadir; Fig. 1B) is increased when the negative effect of strained XBs on the state of other force-bearing XBs is more pronounced. An increase in γ suggests that more force-bearing XBs detach from the thin filament. Because XBs do not interact directly with other XBs, this effect of strained XBs on other force-bearing XBs is transmitted along the thin filament via nonlinear (cooperative) effects. Thus, γ represents the allosteric/cooperative mechanisms by which distorted XBs negatively impact other force-bearing XBs.

b.

Soon after reaching a minimum (nadir), force increases gradually at a characteristic dynamic rate (b) in phase 3 (Fig. 1B). This gradual increase in force is a consequence of recruitment of additional XBs into the force-bearing state corresponding to an increase in ML.

Measurement of k_tr

A modification to the large slack-restretch maneuver, originally described by Brenner and Eisenberg (2), was used to estimate k_tr (pCa 4.3). Once F_ss was achieved, the motor arm was commanded to rapidly slacken the muscle fiber by 10% of the ML using a high-speed length-control device (catalog no. 322C, Aurora Scientific). After a brief (25-ms) shortening period, the motor arm was commanded to rapidly (within 0.5 ms) swing past the original set point by a 10% stretch. The 10% stretch was applied to break any remaining bound XBs. The residual force was not more than ∼10% of the initial F_ss. The rate constant k_tr was determined by fitting the following monoexponential equation to the force redevelopment:

$$\text{F}=\left({\text{F}}_{\text{ss}}-{\text{F}}_{\text{res}}\right)\left(\text{1}-e^{-k_{\mathrm{t}\mathrm{r}}t}\right)+{\text{F}}_{\text{res}}$$

where F is the force at time t and F_res is the residual force from which the redevelopment of force occurs.

Data Analysis

All contractile and mechanodynamic parameters were analyzed using a two-way analysis of variance (ANOVA); one factor in this analysis was TnT (TnT_WT or TnT_F72L), and the other factor was MHC (α-MHC or β-MHC). A significant interaction effect suggested that the functional impact of TnT_F72L was differently altered by α- and β-MHC. When the interaction effect was not significant, we interpreted the main effects due to MHC isoform or TnT variants. To determine the functional impact of MHC isoform or TnT variants on various contractile parameters, post hoc multiple comparisons were made using Fisher's uncorrected least significant differences method. Before analyzing the data, we verified that all the NLRD model-derived parameters followed a normal distribution within each group and that the variance in the parameter data was uniform among groups. The Hill equation was fitted to normalized pCa-tension data to estimate pCa₅₀ and the Hill coefficient (n_H). Values are means ± SE. The criterion for statistical significance was set at P < 0.05.

RESULTS

Assesment of MHC Composition and Phosphorylation Levels of Sarcomeric Proteins in Normal and PTU-Treated Rat Hearts

Protein samples were run on a large 4% SDS gel to assess the MHC composition in normal and PTU-treated rat hearts. A representative gel shown in Fig. 2A confirms that the PTU treatment induces a near-complete shift to β-MHC in rat hearts. We and others previously demonstrated that PTU treatment shifts the MHC isoform from predominantly α-MHC to β-MHC in the ventricles without changing other contractile proteins (18, 25, 34). Previous studies established that the stoichiometry of sarcomeric proteins is maintained in PTU-treated rat cardiac muscle fibers (16, 25, 31, 34, 35). In Fig. 2B, a Pro-Q Diamond-stained gel demonstrates that the phosphorylation levels of proteins (myosin-binding protein C, desmin, TnT, Tm, and myosin light chain-1 and -2) are similar in normal and PTU-treated rat hearts, with one minor exception: a slightly higher level of TnI phosphorylation in PTU-treated than normal rat hearts. However, given that the endogenous Tn is replaced by the recombinant Tn containing the unphosphorylated TnI in our study, small differences in the level of TnI phosphorylation in the PTU-treated group are unlikely to affect the outcome of our study.

Fig. 2.

SDS-PAGE and Western blot analysis of cardiac muscle fibers. A: myosin heavy chain (MHC) isoform composition in normal and propylthiouracil (PTU)-treated rat hearts. Ventricular tissue from normal and PTU-treated rat hearts was solubilized in 2.5% SDS solution, and protein samples were loaded and run on a large 4% SDS gel for optimal separation of MHC isoforms. B: Pro-Q Diamond-stained gel showing phosphorylation status of various sarcomeric proteins in normal and PTU-treated rat hearts. Equal quantities of ventricular protein samples from normal and PTU-treated rat hearts were run on a 12.5% SDS gel. MyBP-C, myosin-binding protein C; Tn, troponin; Tm, tropomyosin; MLC, myosin light chain. C and D: Western blot analysis of mutant TnT incorporation in α-MHC (C) and β-MHC (D) fibers. Reconstituted muscle fibers were run on a small 8% SDS gel. TnT was probed using anti-TnT primary and secondary antibodies. Lane 1, TnT_WT-reconstituted fibers; lane 2, rat TnT analog of the human F70L mutation (TnT_F72L)-reconstituted fibers; lane 3, purified endogenous and c-myc-tagged TnT protein standards.

Western Blot Analysis of the Levels of TnT Incorporation in α- and β-MHC Fibers

We used Western blot analysis to quantify the level of mutant TnT incorporation in reconstituted muscle fibers. In our reconstitution procedure, the endogenous Tn complex is removed as a whole from the thin filaments when muscle fibers are treated with an excess amount of exogenous recombinant Tn (8, 10, 20, 32). We added an 11-amino acid c-myc tag at the very NH₂ terminus of the mutant TnT proteins, the inclusion of which had no effect on the cardiac muscle function (11, 36, 48). Addition of a c-myc tag enabled us to discriminate between the endogenous TnT and recombinant TnT in gels, which allowed us to determine the level of protein incorporation in myofilaments. Representative Western blots demonstrating the levels of recombinant TnT incorporation in α- and β-MHC fibers are shown in Fig. 2, C and D, respectively. Densitometric analysis of TnT band profiles demonstrated that incorporation of TnT_WT in α-MHC fibers was 76% (Fig. 2C, lane 1), while incorporation of TnT_F72L was 81% (Fig. 2C, lane 2). Similarly, the incorporation levels of TnT_WT and TnT_F72L in β-MHC fibers were 78% (Fig. 2D, lane 1) and 75% (Fig. 2D, lane 2), respectively. Uniform levels of TnT_F72L incorporation in α- and β-MHC fibers provided a good experimental model to test how the interplay between TnT- and MHC-mediated effects on the thin filament altered contractile function differently.

Impact of TnT_F72L on Ca²⁺-Activated Maximal Tension and E_D in α- and β-MHC Fibers

To determine whether TnT_F72L differentially altered maximal activation of thin filaments in α- and β-MHC fibers, we first assessed the Ca²⁺-activated maximal tension. Comparison of experimental force records during isometric contraction at pCa 4.3 suggests that TnT_F72L decreases maximal tension in α-MHC fibers (Fig. 3A), while it increases tension in β-MHC fibers (Fig. 3B). Two-way ANOVA revealed a significant MHC-TnT interaction effect on maximal tension (P < 0.01), suggesting that the effect of TnT_F72L varied with the MHC isoform. Post hoc tests confirmed that TnT_F72L significantly decreased maximal tension by 15% in α-MHC fibers (P < 0.05; Fig. 3C), whereas it increased tension by 10% in β-MHC fibers (P < 0.05; Fig. 3C). The divergent impact of TnT_F72L on tension is likely related to differences in the number of strongly bound XBs in α- and β-MHC fibers. To determine whether this is the case, we analyzed the statistical differences in E_D between fiber groups. Two-way ANOVA confirmed a significant MHC-TnT interaction effect on E_D (P < 0.01), suggesting that the effect of TnT_F72L on E_D was altered differently by α- and β-MHC isoforms. Post hoc analysis confirmed that TnT_F72L significantly attenuated E_D by 20% in α-MHC fibers (P < 0.05; Fig. 3D), whereas TnT_F72L increased E_D by 16% in β-MHC fibers (P < 0.05; Fig. 3D). Because the effects on tension are corroborated by E_D, our findings suggest that the TnT_F72L-mediated effects on the thin filament interact differently with those induced by α- and β-MHC to bring about contrasting effects on the number of strongly bound XBs.

Fig. 3.

Effects of TnT_F72L on Ca²⁺-activated maximal tension and instantaneous muscle fiber stiffness caused by a sudden change in ML (E_D) in α- and β-MHC fibers. A and B: effect of TnT_F72L on isometric tension at pCa 4.3 in α- and β-MHC fibers. C and D: differential impact of TnT_F72L on maximal tension and E_D in α- and β-MHC fibers. E_D was estimated by fitting a linear regression line to the relationship between F₁ − F_ss (see Fig. 1B) and the corresponding ML changes (ΔL). TnT_WT, wild-type rat TnT. Values are means ± SE; n = 11 fibers for α-MHC + TnT_WT (2 hearts), 13 for α-MHC + TnT_F72L (2 hearts), 13 for β-MHC + TnT_WT (2 hearts), and 14 for β-MHC + TnT_F72L (2 hearts). Statistical differences were analyzed by 2-way ANOVA and subsequent post hoc multiple pair-wise comparisons (Fisher's least significant differences method): *P < 0.05 vs. TnT_WT.

Impact of TnT_F72L on pCa-Tension Relationships in α- and β-MHC Fibers

The effect of TnT_F72L on the pCa-tension relationship was assessed using pCa₅₀ (a measure of myofilament Ca²⁺ sensitivity) and n_H (a measure of myofilament cooperativity), both of which are obtained by fitting the Hill model to pCa-tension relationships. A comparison of Hill model fits showed that TnT_F72L induced a rightward shift of the pCa-tension relationship in α-MHC fibers (Fig. 4A), suggesting a decrease in myofilament Ca²⁺ sensitivity. In contrast, TnT_F72L induced a leftward shift in β-MHC fibers (Fig. 4B), suggesting an increase in myofilament Ca²⁺ sensitivity. Two-way ANOVA of pCa₅₀ revealed a significant MHC-TnT interaction effect (P < 0.01), confirming that the effect of TnT_F72L on pCa₅₀ was altered differently by α- and β-MHC. Post hoc analysis confirmed that TnT_F72L decreased pCa₅₀ by 0.06 pCa unit in α-MHC fibers (P < 0.01; Fig. 4C), whereas it increased pCa₅₀ by 0.05 pCa unit in β-MHC fibers (P < 0.01; Fig. 4C). Because small, but significant, changes in pCa₅₀ are difficult to appreciate, we compared the steady-state tension data of different fiber groups at pCa 5.5. TnT_F72L attenuated tension to a greater extent (i.e., 29% at pCa 5.5 vs. 15% at pCa 4.3) in α-MHC fibers, while it had an opposite effect (i.e., an increase of 25% at pCa 5.5 vs. 10% at pCa 4.3) in β-MHC fibers. Therefore, at submaximal Ca²⁺ concentrations (conditions under which cardiac muscles normally operate), the magnitude of TnT_F72L-mediated attenuation of tension is greater in α-MHC fibers and the magnitude of TnT_F72L-mediated augmentation of tension is greater in β-MHC fibers. These observations suggest that TnT_F72L- and α-MHC-induced changes in the thin filament interact to desensitize cardiac thin filaments to Ca²⁺, whereas those induced by TnT_F72L and β-MHC sensitize cardiac thin filaments to Ca²⁺.

Fig. 4.

Effects of TnT_F72L on the pCa-tension relationship in α- and β-MHC fibers. Normalized tension values were plotted against the respective pCa values to construct pCa-tension relationships. A Hill's model was fitted to pCa-tension relationships to derive pCa₅₀ (myofilament Ca²⁺ sensitivity) and Hill's coefficient [n_H (myofilament cooperativity)]. A and B: effect of TnT_F72L on the pCa-tension relationship in α- and β-MHC fibers. C and D: differential impact of TnT_F72L on pCa₅₀ and n_H in α- and β-MHC fibers. Values are means ± SE; n = 11 fibers for α-MHC + TnT_WT (2 hearts), 13 for α-MHC + TnT_F72L (2 hearts), 13 for β-MHC + TnT_WT (2 hearts), and 14 for β-MHC + TnT_F72L (2 hearts). Statistical differences were analyzed by 2-way ANOVA and subsequent post hoc multiple pair-wise comparisons (Fisher's least significant differences method): *P < 0.05, **P < 0.01 vs. TnT_WT. NS, not significant.

A cursory look at pCa-tension relationships also suggests that TnT_F72L decreases the steepness of the pCa-tension relationship in α-MHC fibers (Fig. 4A) but shows no effect in β-MHC fibers (Fig. 4B). Two-way ANOVA showed a significant MHC-TnT interaction effect on n_H (P < 0.05). Post hoc tests revealed that TnT_F72L significantly decreased n_H by 22% in α-MHC fibers (P < 0.01; Fig. 4D) but showed no effect in β-MHC fibers (P = 0.81; Fig. 4D). These observations suggest that the TnT_F72L- and α-MHC-induced changes in the thin filament interact to attenuate myofilament cooperativity.

Impact of TnT_F72L on the Ca²⁺-Activated Maximal ATPase Activity in α- and β-MHC Fibers

To determine if changes in tension corresponded to changes in ATPase activity, we analyzed ATPase activity between fiber groups. Two-way ANOVA did not show a significant MHC-TnT interaction effect (P = 0.41) on ATPase activity. The main effect was not significant (P = 0.62), because the post hoc analysis showed that TnT_F72L did not alter ATPase activity in α-MHC (P = 0.44) or β-MHC (P = 0.87) fibers. The estimates of ATPase activity (in pmol·mm⁻³·s⁻¹) in various groups are as follows: 154 ± 6 (n = 11) for α-MHC + TnT_WT, 163 ± 11 (n = 13) for α-MHC + TnT_F72L, 95 ± 7 (n = 13) for β-MHC + TnT_WT, and 104 ± 6 (n = 14) for β-MHC + TnT_F72L. Thus, unlike tension, the TnT_F72L-mediated effect on ATPase activity was not altered differently by α- and β-MHC. However, lower tension, but normal ATPase activity, in α-MHC + TnT_F72L fibers suggested that TnT_F72L- and α-MHC-induced changes in the thin filament could interact to augment XB detachment rate (Fig. 5).

Fig. 5.

A and B: effects of TnT_F72L on tension cost and c in α- and β-MHC fibers. Tension cost was estimated as the slope of the linear regression between steady-state tension and ATPase measurements at various levels of pCa (14, 47); c was estimated by fitting the nonlinear recruitment distortion model to the family of fiber-elicited force responses to steplike ML perturbations at pCa 4.3 (17). Values are means ± SE; n = 11 fibers for α-MHC + TnT_WT (2 hearts), 13 for α-MHC + TnT_F72L (2 hearts), 13 for β-MHC + TnT_WT (2 hearts), and 14 for β-MHC + TnT_F72L (2 hearts). Statistical differences were analyzed by 2-way ANOVA and subsequent post hoc multiple pair-wise comparisons (Fisher's least significant differences method): **P < 0.01, ***P < 0.001 vs. TnT_WT. NS, not significant.

Impact of TnT_F72L on XB Detachment Rate in α- and β-MHC Fibers

Previous studies have shown that TnT alters XB detachment rate (g); this effect is MHC isoform-dependent (18, 22, 35, 50). Therefore, we analyzed how the TnT_F72L-mediated effects on tension cost (ATPase/tension) and the rate constant of XB distortion dynamics (c) are altered differently by α- and β-MHC. Tension cost is an approximation of g, because the ratio of ATPase (fg/f + g) to tension (f/f + g) is proportional to g (1, 13, 22), where f and g are XB attachment and detachment rates, respectively. Tension cost was estimated as the slope of the linear regression between the steady-state tension and ATPase values measured at various pCa levels (14, 33, 47). The parameter c governs the rate of force decay following a steplike ML change and is estimated by fitting the NLRD model to the family of force responses to various-amplitude stretch-release perturbations at pCa 4.3 (17). Previous studies have demonstrated that tension cost and c show a positive correlation and that both are useful measures of g (4). Two-way ANOVA showed a significant MHC-TnT interaction effect on tension cost (P < 0.05), suggesting that the effect of TnT_F72L on tension cost was significantly different in α- and β-MHC fibers. Post hoc analysis demonstrated that TnT_F72L increased tension cost by 33% in α-MHC fibers (P < 0.001; Fig. 5A) but had no effect in β-MHC fibers (P = 0.57; Fig. 5A). In agreement with our tension cost data, two-way ANOVA of c showed a significant MHC-TnT interaction effect (P < 0.001), suggesting that the effect of TnT_F72L on c was differently altered by α- and β-MHC isoforms. Post hoc tests confirmed that TnT_F72L increased c by 49% in α-MHC fibers (P < 0.001; Fig. 5B) but showed no effect in β-MHC fibers (P = 0.69; Fig. 5B). Similar changes in tension cost and c provided evidence substantiating the speeding effect of TnT_F72L on g in α-MHC, but not β-MHC, fibers.

Impact of TnT_F72L on XB Turnover Rates in α- and β-MHC Fibers

The rate constants of tension redevelopment (k_tr) and XB recruitment dynamics (b) were measured in muscle fibers at pCa 4.3 to validate whether α- and β-MHC differently altered XB turnover rate in TnT_F72L fibers. The parameter k_tr represents the rate constant of force recovery to the steady state following a large release-restretch perturbation (2). Similarly, b represents the rate of delayed force rise following a steplike ML change (Fig. 1B) and is estimated by fitting the NLRD model to a family of force responses to various-amplitude stretch-release perturbations (17). We previously demonstrated that k_tr is strongly correlated to b and that both are useful measures of XB turnover rates (4, 20, 22). Examination of the experimental force responses to a large release-restretch length perturbation suggests that TnT_F72L augments the rate of force recovery in α-MHC fibers (Fig. 6A), whereas it attenuates this rate in β-MHC fibers (Fig. 6B). Two-way ANOVA revealed a significant MHC-TnT interaction effect on k_tr (P < 0.001), confirming divergent effects of TnT_F72L on k_tr in α- and β-MHC fibers. Post hoc tests confirmed that TnT_F72L augmented k_tr by 28% in α-MHC fibers (P < 0.001; Fig. 6C), while TnT_F72L attenuated k_tr by 15% in β-MHC fibers (P < 0.01; Fig. 6C). Estimates of b also showed similar results. For instance, two-way ANOVA showed a significant MHC-TnT interaction effect on b (P < 0.001), confirming that the TnT_F72L-mediated effect on b was significantly different in α- and β-MHC fibers. Post hoc tests confirmed that TnT_F72L increased b by 22% in α-MHC fibers (P < 0.001; Fig. 6D), while TnT_F72L attenuated b by 15% in β-MHC fibers (P < 0.05; Fig. 6D). A faster b suggested that TnT_F72L led to an increase in the rate of newly recruited strong XBs in α-MHC fibers. Conversely, a slower b suggested that TnT_F72L led to a decrease in the rate of recruitment of new XBs in β-MHC fibers. Similar effects on b and k_tr suggest that TnT_F72L-induced changes in the thin filament interact differently with changes induced by α- and β-MHC to bring about contrasting effects on XB turnover rates.

Fig. 6.

Effects of TnT_F72L on b and k_tr in α- and β-MHC fibers. The rate at which new force-bearing XBs are recruited following a quick steplike change in ML (b) (17) and k_tr was determined as described in methods. A and B: effect of TnT_F72L on the force response to a large release-restretch perturbation in α- and β-MHC fibers. Forces were normalized by the new isometric steady-state force attained after length perturbation. C and D: differential impacts of TnT_F72L on k_tr and b in α- and β-MHC fibers. Values are means ± SE; n = 11 for α-MHC + TnT_WT (2 hearts), 13 for α-MHC + TnT_F72L (2 hearts), 13 for β-MHC + TnT_WT (2 hearts), and 14 for β-MHC + TnT_F72L (2 hearts). Statistical differences were analyzed by 2-way ANOVA and subsequent post hoc multiple pair-wise comparisons (Fisher's least significant differences method): *P < 0.05, ***P < 0.001 vs. TnT_WT.

Different Effects of TnT_F72L on γ in α- and β-MHC Fibers

Novel insights into how the TnT_F72L-mediated impact on thin filament cooperativity is differentially modified by α- and β-MHC may be gleaned from comparisons of γ, because it represents nonlinear effects (cooperativity) by which strained XBs negatively impact other force-bearing XBs (16, 17, 22). The greater the γ, the larger is the force decline to a minimum force point (i.e., more prominent nadir; Fig. 1B). Therefore, an increase in γ suggests that the negative impact of strained XBs on force-bearing XBs becomes more prominent when the thin filament cooperativity is altered (16, 17, 22). A comparison of force responses to 2% quick stretch demonstrates that TnT_F72L causes a more prominent nadir in α-MHC fibers (Fig, 7A) but shows no effect in β-MHC fibers (Fig. 7B). Two-way ANOVA showed a significant MHC-TnT interaction effect on γ (P < 0.05), confirming that the effect of TnT_F72L on γ was modulated differently by α- and β-MHC. Post hoc analysis revealed that TnT_F72L increased γ by 39% in α-MHC fibers (P < 0.001; Fig. 7C), whereas TnT_F72L showed no effect in β-MHC fibers (P = 0.88; Fig. 7C). The attenuating effect of TnT_F72L on myofilament cooperativity (Fig. 4D) increased the negative impact of strained XBs on other force-bearing XBs; hence, γ increased significantly in α-MHC fibers.

Fig. 7.

Effect of TnT_F72L on γ [the cooperative mechanisms by which distorted XBs negatively impact other force-bearing XBs (17)] in α- and β-MHC fibers. Changes in γ reflect alterations in cooperativity operating within the thin filament; γ was determined as described in methods. A and B: effect of TnT_F72L on the force response to a 2% quick stretch perturbation in α- and β-MHC fibers. Forces were normalized by the isometric steady-state force just prior to the stretch perturbation. C: differential impact of TnT_F72L on γ in α- and β-MHC fibers. Values are means ± SE; n = 11 fibers for α-MHC + TnT_WT (2 hearts), 13 for α-MHC + TnT_F72L (2 hearts), 13 for β-MHC + TnT_WT (2 hearts), and 14 for β-MHC + TnT_F72L (2 hearts). Statistical differences were analyzed by 2-way ANOVA and subsequent post hoc multiple pair-wise comparisons (Fisher's least significant differences method): ***P < 0.001 vs. TnT_WT. NS, not significant.

DISCUSSION

Our observation that α- and β-MHC isoforms divergently modify the activity of NTE in TnT (31) has significant implications for human heart disease, because three different HCM-related mutations are located in the NTE (28, 39, 42). The molecular mechanisms by which these mutations affect cardiac contractile function have not been studied. In this study we provide new evidence for the emerging paradigm of disparate effects of α- and β-MHC on cardiomyopathy-related mutations in TnT. Novel findings from our study suggest contrasting effects of TnT_F72L on cardiac contractile dysfunction in the presence of α- and β-MHC. The mechanistic implications of our study are discussed in terms of the emergence of divergent thin filament cooperativity and their disparate effects on cardiac contractile function.

TnT_F72L-Mediated Effects on Tension Are Divergently Modified by α- and β-MHC

An important finding from our study was that TnT_F72L significantly decreased Ca²⁺-activated maximal tension in the presence of α-MHC but increased tension in the presence of β-MHC (Fig. 3C). To address how the TnT_F72L-mediated effects are divergently expressed in α- and β-MHC fibers, we must first consider the location of the mutation and the characteristics of its flanking regions. TnT_F72L is located in the NTE of TnT and is adjacent to the CR of TnT. Findings from our recent study (20) suggest that 1) the CR modulates XB recruitment dynamics via its effect on thin filament cooperativity and 2) the NTE has a synergistic effect on the ability of the CR to modulate XB recruitment dynamics. Because of the close proximity of this mutation to the CR of TnT, the effect of mutation-induced local structural changes may spread to the CR to alter its interaction with Tm and XB recruitment dynamics. Altered CR-Tm interaction has been suggested to modify the number of Ca²⁺-activated RUs and tension (20, 38, 45, 49). Activation of RUs is critical for XB entry into the cycling pool; therefore, attenuation of RU activation is expected to decrease the number of strongly bound XBs in the cycling pool. Because E_D is a measure of the number of strong XBs in the steady state (17), the TnT_F72L-induced attenuation of E_D in α-MHC fibers (Fig. 3D) confirms that the F72L mutation decreases the number of strong XBs and tension. Reduction of the number of strong XBs leads to attenuation of XB-RU- and XB-XB-based cooperativity (23, 37). The TnT_F72L-mediated attenuation of XB-RU- and XB-XB-based cooperativity in α-MHC fibers caused a significant decrease in myofilament Ca²⁺ sensitivity (Fig. 4C) and myofilament cooperativity (Fig. 4D). We expect a higher level of XB-RU- and XB-XB-based cooperativity in β-MHC fibers because of the longer dwell time of β-MHC XBs in the strongly bound state (16, 44, 46). Such β-MHC-enhanced XB-based cooperativity is expected to promote thin filament activation in TnT_F72L fibers. This is consistent with increases in maximal tension (Fig. 3C), E_D (Fig. 3D), myofilament Ca²⁺ sensitivity (Fig. 4C), and restoration of myofilament cooperativity to normal levels (Fig. 4D) in TnT_F72L + β-MHC fibers. Collectively, these observations demonstrate that TnT_F72L attenuates thin filament cooperativity in α-MHC, but not β-MHC, fibers because of differential impacts of α- and β-MHC on the thin filament. This assertion is supported by our recent observation that β-MHC affects the transition between off/on states of the RU when the NTE is altered (31).

TnT_F72L-Mediated Effects on XB Recruitment Dynamics Are Divergently Modified by α- and β-MHC

Strong dependence of contractile dynamics on cooperativity (3, 4, 41) suggests that the TnT_F72L-mediated effects on thin filament cooperativity and XB recruitment dynamics are differently altered by α- and β-MHC. A decrease in XB-XB-based cooperativity increases the speed of XB recruitment; conversely, an increase in XB-XB-based cooperativity decreases the speed of XB recruitment (3, 41). Shorter dwell time of strong α-MHC XBs is expected to decrease cooperativity, and longer dwell time of strong β-MHC XBs is expected to increase cooperativity (22, 44, 46). What follows from this argument is that the TnT_F72L-induced attenuation of XBs has an exacerbating effect on lower cooperativity in α-MHC fibers, leading to an increase in the rate of XB recruitment. Conversely, the augmenting effect of β-MHC on cooperativity overcomes the negative effect of TnT_F72L on cooperativity, leading to a decrease in the rate of XB recruitment. Just as we predicted, b increased in α-MHC + TnT_F72L fibers but decreased in β-MHC + TnT_F72L fibers (Fig. 6D). Similarly, k_tr increased in α-MHC + TnT_F72L fibers but decreased in β-MHC + TnT_F72L fibers (Fig. 6C). Enhanced XB-RU/XB-XB cooperativity, as in the case of β-MHC, is also expected to increase the magnitude of activation (6, 41). This assertion is substantiated by increases in maximal tension (Fig. 3C), E_D (Fig. 3D), and pCa₅₀ (Fig. 4C) in β-MHC + TnT_F72L fibers.

Effect of TnT_F72L on XB Detachment Kinetics Is Differently Modulated by α- and β-MHC

Effects on tension cost (Fig. 5A) and c (Fig. 5B) confirmed that TnT_F72L increased the rate of XB detachment kinetics (g) in α-MHC fibers; however, TnT_F72L did not have an effect on g in β-MHC fibers. An increase in g shifts the equilibrium of the XB population more toward the non-force-bearing state in α-MHC + TnT_F72L fibers. This would explain why maximal tension and E_D are significantly lower in α-MHC + TnT_F72L fibers, because, according to the two-state XB model (26), the fraction of attached XBs in the isometric steady state is proportional to f/(f + g). The augmenting effect of TnT_F72L on g shortens the dwell time of α-MHC XBs in the strongly bound state, leading to attenuation of the XB-based feedback effect on the thin filament. Our interpretations on the effects of α-MHC on g are substantiated by significant decreases in maximal tension (Fig. 3C), E_D (Fig. 3D), pCa₅₀ (Fig. 4C), and n_H (Fig. 4D) of α-MHC + TnT_F72L fibers. On the other hand, the TnT_F72L-mediated increase in g is counteracted by the slower kinetics of β-MHC XBs; therefore, g remains unaltered in β-MHC + TnT_F72L fibers (Fig. 5). The longer duty cycle of β-MHC XBs not only ablates the augmenting effect of TnT_F72L on g, but it also promotes thin filament activation in TnT_F72L fibers because of increased XB-XB cooperativity. This would shift the equilibrium of the XB population more toward the force-bearing state in β-MHC + TnT_F72L fibers. Our observations on the β-MHC effect are consistent with increases in maximal tension (Fig. 3C), E_D (Fig. 3D), and myofilament Ca²⁺ sensitivity (Fig. 4C). Once again, our data demonstrate that the TnT_F72L-mediated effects on the thin filament are differently modulated by α- and β-MHC.

Impact of TnT_F72L on γ Is Differently Modulated by α- and β-MHC

The parameter γ represents nonlinear effects (cooperativity) in the thin filament whereby strained XBs negatively impact other force-bearing XBs (17). Changes in γ reflect changes in cooperativity operating within the thin filament. TnT_F72L increases γ in the presence of α-MHC (Fig. 7C), which suggests that the negative impact of strained XBs on other force-bearing XBs is more pronounced in α-MHC + TnT_F72L fibers. We believe that TnT_F72L exerts its effect via its inhibitory actions on RUs, which leads to a decrease in the number of strong XBs and subsequent attenuation of XB-RU/XB-XB cooperativity. The negative impact of strained XBs on force is more pronounced in α-MHC + TnT_F72L fibers, because the cooperativity at the onset of ML change is lower due to the attenuating effect of TnT_F72L on thin filament cooperativity. Such actions of TnT_F72L eventually lead to a greater γ in α-MHC + TnT_F72L fibers. Conclusions drawn from estimates of γ validate our findings from the pCa-tension relationship, which demonstrate that TnT_F72L attenuates myofilament cooperativity in α-MHC + TnT_F72L fibers (Fig. 4D). TnT_F72L has no effect on γ in β-MHC fibers, because when the positive effect of β-MHC on cooperativity is imposed on the negative effect of TnT_F72L on cooperativity, γ remains unaltered. Therefore, the cooperativity at the onset of ML change is expected to be normal in β-MHC + TnT_F72L fibers. Here again, conclusions drawn from estimates of γ substantiate our findings from the pCa-tension relationship, which showed that the attenuating effect of TnT_F72L on cooperativity is ablated by β-MHC (Fig. 4D). Collectively, two independent experimental observations (pCa-tension relationships and estimates of γ) substantiate our finding that the TnT_F72L-mediated impact on thin filament cooperativity is differently modulated by α- and β-MHC.

Summary and Conclusion

Molecular mechanisms by which the F70L mutation in human TnT affects cardiac contractile function have not been studied. TnT_F72L-mediated effects on tension, myofilament Ca²⁺ sensitivity, myofilament cooperativity, rates of tension redevelopment, XB detachment kinetics, and XB recruitment dynamics were divergently modified by α- and β-MHC. The mechanistic basis of our finding is that the TnT_F72L-mediated effects on thin filament cooperativity are differently affected by α- and β-MHC. More importantly, maximal tension and myofilament Ca²⁺ sensitivity were attenuated in TnT_F72L + α-MHC fibers but augmented in TnT_F72L + β-MHC fibers. Previous studies established decreased myofilament Ca²⁺ sensitivity (i.e., depressed contractility) and systolic dysfunction as hallmarks of dilated cardiomyopathy-related mutations, whereas increased myofilament Ca²⁺ sensitivity (i.e., enhanced contractility) and diastolic dysfunction as hallmarks of HCM-related mutations (reviewed in Refs. 29, 51, and 52). These findings, when aligned with those from our study, suggest that TnT_F72L induces a dilated cardiomyopathy-like phenotype in the presence of α-MHC. On the other hand, TnT_F72L mimics a HCM-like phenotype in the presence of β-MHC, a result that is consistent with the clinical data in human hearts (42). When increases in Ca²⁺ sensitivity (hypercontractility) and XB-XB/XB-RU cooperativity, as observed in β-MHC + TnT_F72L fibers, are extrapolated to the human heart, one may expect a significant increase in tension at the free Ca²⁺ concentration at which the heart normally operates. Indeed, our results show that the augmentation of tension is more pronounced at submaximal free Ca²⁺ concentration in β-MHC + TnT_F72L fibers. Such an increase in tension may lead to diastolic dysfunction, because the myocardial relaxation is load-dependent. Finally, our data demonstrate that inferences drawn from studies of rodent hearts (expressing α-MHC) must be viewed with caution because of differences in MHC isoform composition between human and rodent hearts.

GRANTS

This work was funded by National Heart, Lung, and Blood Institute Grant R01-HL-075643 to M. Chandra.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

V.C., S.K.G., and M.C. developed the concept and designed the research; V.C. and S.K.G. performed the experiments; V.C. and S.K.G. analyzed the data; V.C., S.K.G., and M.C. interpreted the results of the experiments; V.C. and S.K.G. prepared the figures; V.C., S.K.G., and M.C. drafted the manuscript.