Familial hypertrophic cardiomyopathy mutations in troponin I (K183
, G203S, K206Q) enhance filament sliding1
Jan Köhler1,
Ying Chen2,
Bernhard Brenner1,
Albert M. Gordon3,
Theresia Kraft1,
Donald A. Martyn4,
Michael Regnier4,
Anthony J. Rivera4,
Chien-Kao Wang3,4 and
P. Bryant Chase5
1 Molekular- und Zellphysiologie, Medizinische Hochschule, D-30625 Hannover, Germany
2 Departments of Radiology
3 Physiology and Biophysics
4 Bioengineering, University of Washington, Seattle, Washington
5 Department of Biological Science, Florida State University, Tallahassee, Florida 32306-4370
 |
ABSTRACT
|
---|
A major cause of familial hypertrophic cardiomyopathy (FHC) is dominant mutations in cardiac sarcomeric genes. Linkage studies identified FHC-related mutations in the COOH terminus of cardiac troponin I (cTnI), a region with unknown function in Ca2+ regulation of the heart. Using in vitro assays with recombinant rat troponin subunits, we tested the hypothesis that mutations K183
, G203S, and K206Q in cTnI affect Ca2+ regulation. All three mutants enhanced Ca2+ sensitivity and maximum speed (smax) of filament sliding of in vitro motility assays. Enhanced smax (pCa 5) was observed with rabbit skeletal and rat cardiac (
-MHC or ß-MHC) heavy meromyosin (HMM). We developed a passive exchange method for replacing endogenous cTn in permeabilized rat cardiac trabeculae. Ca2+ sensitivity and maximum isometric force did not differ between preparations exchanged with cTn(cTnI,K206Q) or wild-type cTn. In both trabeculae and motility assays, there was no loss of inhibition at pCa 9. These results are consistent with COOH terminus of TnI modulating actomyosin kinetics during unloaded sliding, but not during isometric force generation, and implicate enhanced cross-bridge cycling in the cTnI-related pathway(s) to hypertrophy.
heart; calcium regulation; systole; in vitro motility; troponin exchange
 |
INTRODUCTION
|
---|
FAMILIAL HYPERTROPHIC CARDIOMYOPATHY (FHC) is a disease caused by dominant mutations in genes for proteins of the cardiac sarcomere (4, 38, 39, 48, 55, 68). It is typically characterized by ventricular hypertrophy, often resulting in arrhythmias and possibly sudden cardiac death (SCD). The extent of hypertrophy and prognosis for affected individuals varies widely and depends on the specific mutation involved and other less well-defined factors. Tragically, SCD may be the first manifestation in young, seemingly fit individuals.
Kimura et al. (31) reported genetic linkage studies identifying mutations in the cardiac troponin I (cTnI) gene that lead to FHC. Their initial study identified six mutations in five residues, all in the COOH-terminal third of cTnI (exons 7 and 8). Subsequent reports have identified four additional FHC-related mutations in cTnI, all but one of which are located near the COOH terminus (43, 44, 47). Troponin I (I = inhibitory) is crucial for turning myofilaments "off" when intracellular [Ca2+] is low during diastole (21, 48). Central to this function is the inhibitory peptide region of cTnI in which two mutations were identified at residue R145 (31). Recent work has established that the R145G variant has functional effects consistent with early peptide studies by Van Eyk and Hodges (70) and also the hypertrophic phenotype (9, 19, 29, 35, 67). Although considerable effort has gone into studies of the R145G mutation and structure of the inhibitory region, only limited work has been reported on the other mutations in cTnI, and little is known about the structure of the COOH-terminal region. The work of Takahashi-Yanaga et al. (67) showed that all but the G203S mutation increased Ca2+ sensitivity of ATPase activity and isometric force production, although the reported changes with the K206Q mutation were small. These results contrasted with Burton et al. (9), who showed that G203S enhanced Ca2+ sensitivity of force generation but not ATPase activity, whereas R145G did not affect Ca2+ sensitivity of force. Both Takahashi-Yanaga et al. (67) and Burton et al. (9), and additionally Lang et al. (35), agree that the R145 mutations resulted in significant Ca2+-independent force. Studies that examined isometric force generation, however, incorporated recombinant cTnI into the myofilament lattice using procedures that involve prolonged activation during extraction/reconstitution, which can result in significant decreases in maximum force and/or increases in force at "relaxing" Ca2+ levels with wild-type (WT) cTnI. In one other study, Elliott et al. (19) showed that the R162W mutation increased both Ca2+ sensitivity of actin-Tn-Tm-S1-ATPase activity in solution and also ATPase at very low [Ca2+].
Because only limited and in some instances contradictory information is currently available, we sought to identify changes in myofilament function caused by the most COOH-terminal FHC-related mutations in cTnI which would enable us to infer a mechanism(s) by which these mutations lead to cardiac hypertrophy. Cardiac troponin subunits from rat, WT or with site-directed mutations equivalent to those found in FHC, were expressed in bacterial systems. Recombinant rat cTns were evaluated using Ca2+-regulated in vitro motility assays to quantitatively examine the sliding of individual actin filaments. A procedure was also developed for exchanging the recombinant cTns into permeabilized cardiac ventricular trabeculae to examine effects of mutations on force generation. Our results suggest that the K183
, G203S, and K206Q mutations enhance filament sliding and thus may cause hypertrophy via a signaling pathway that responds to increased systolic ATPase activity in cardiomyocytes.
Portions of this work have been published in abstract form (14, 15).
 |
METHODS
|
---|
Protein Preparations
Troponin subunits.
Cloning and expression of rat cardiac WT cTnC was previously described (17). Rat cardiac WT cTnI and WT cTnT were similarly cloned from total RNA isolated from adult rat cardiac muscle by the guanidium isothiocyanate method of Chomczynski and Sacchi (16). Mutations were introduced at rat cTnI residues equivalent to human cTnI residues K183
(codon deletion), G203S or K206Q and also at the x position of the low-affinity, NH2-terminal Ca2+ binding site II, cTnC (D65A) (also referred to as xcTnC), by site-directed mutagenesis using the T7-GEN In Vitro Mutagenesis Kit (US Biochemicals, Cleveland, OH). A vector pET-24 (Novagen, Madison, WI) containing the T7 promoter, lac operator, and a kanamycin resistance gene was used for the expression of the respective WT or mutant clones in Escherichia coli, and the protein was extracted from bacterial cells as described for rat cardiac cTnC (17) and purified according to methods described for native cTnC, cTnI, or cTnT (52). Although the COOH-terminal residue numbering is different between rat and human cTnI because of the additional residue A24 in the NH2 terminus of rat cTnI, we retain the nomenclature of the human sequence to avoid confusion with clinical literature. For a limited set of control studies, bovine cTnT was purified from fresh heart tissue obtained from a local abattoir according to previously described methods (52). Concentration and purity of troponin subunits and all other proteins described below were evaluated by UV absorbance and SDS-PAGE, respectively (20).
Rhodamine labeling of cTnT and xcTnC.
The fluorescent probe, the 5' isomer of iodoacetamidotetramethylrhodamine (5'-IATR), was a generous gift of Dr. John Corrie (National Institute for Medical Research, Mill Hill, London). The recombinant cTnC mutant, xcTnC, was labeled with 5'-IATR under denaturing conditions as previously described for sTnC (41). Native bovine cTnT was similarly labeled at Cys39 under denaturing conditions. Labeling was 0.2 mol of rhodamine per mol of xcTnC or 0.4 mol of rhodamine per mol of cTnT.
Troponin complex.
cTn was purified from frozen rat hearts (Pel-Freez, Rogers, AZ) as described for bovine heart (52). Cardiac Tn complex was reconstituted from isolated (recombinant or native) subunits (1:1:1 ratio) as described for native Tn subunits (52).
Myosin, actin, and tropomyosin.
Skeletal myosin and heavy meromyosin (sHMM) were prepared from rabbit back and leg muscles as described previously (13, 20). Myosin was stored in 50% vol/vol glycerol at -20°C for up to 6 wks; sHMM was stored at 04°C for up to 1 wk. Cardiac myosin and HMM (cHMM) were prepared as previously described (57) from control rat hearts or from rats treated with 0.8 mg/ml propylthiouracil (PTU) in their drinking water. Freshly prepared cardiac myosin was used immediately to make cHMM, and cHMM was used within 3 days of preparation. Cardiac myosin and HMM were stored at 04°C. At the beginning of each day of motility experiments, ATP-insensitive heads were removed from an aliquot of HMM by ultracentrifugation (20, 34). A 1.5-fold molar excess of F-actin was added to HMM, followed by 1 mM ATP and ultracentrifugation at 513,000 g and 4°C (model TLX 120.2; Beckman, Fullerton, CA). Competent HMM (supernatant) was then diluted to 250 µg/ml (determined using the Bradford assay due to the presence of ATP and ADP), the concentration used in motility experiments. Actin and tropomyosin (Tm) were prepared from rabbit skeletal muscle ether powder as previously described (13, 20, 57) using the methods of Pardee and Spudich (50) and Smillie (62), respectively. F-actin was labeled with rhodamine-phalloidin (RhPh) as described by Kron et al. (34) for visualization by fluorescence microscopy.
In vitro motility assay.
In vitro motility assays were carried out with regulated actin as previously described (20, 56) with minor modifications. Fundamental aspects of the motility assay with rabbit skeletal HMM (13, 20, 56) or rat cardiac HMM (57) and data analysis were performed according to established procedures in our laboratory. Glass microscope slides and no. 1 thickness coverslips were cleaned by sonication in 1 mM KOH and then rinsed, sonicated in, and rinsed again with deionized H2O, and finally oven dried. The coverslips were coated on one side with a thin layer of 0.1% nitrocellulose in amyl acetate (Ernest Fullam, Latham, NY). Flow cells were constructed on microscope slides by placing the nitrocellulose-coated coverslips on no. 1
thickness glass spacers with silicone grease (34). After the chamber was readied, the flow cell was completed by infusing a series of solutions (
2x chamber volume, each), the majority of which were left for 1 min in the chamber and then flushed with actin buffer (AB) made without ATP (25 mM KCl, 25 mM imidazole, 4 mM MgCl2, 1 mM EGTA, 1 mM DTT, pH 7.4) (34) before infusing the next solution. All solutions were allowed to equilibrate to room temperature before infusion into the flow cell to minimize formation of bubbles. HMM was applied to the flow cell first for 1 min. HMM application was immediately followed by 0.5 mg/ml BSA in AB to block nonspecific protein binding. After the chamber was flushed with AB, unlabeled F-actin (
100 µg/ml, sheared by at least 15 rapid passages through a 23-gauge needle) was added. Unbound F-actin was flushed out of the flow cell with AB, then AB with 0.5 mM ATP was added to dissociate remaining unlabeled F-actin from competent HMM on the nitrocellulose-coated surface, thus leaving residual "dead heads" blocked by unlabeled F-actin (34, 59). After again flushing the chamber with AB, 8 nM RhPh-labeled F-actin was applied in the absence of ATP. Labeled actin filaments that did not bind to HMM on the surface were flushed from the chamber with a "wash buffer" that was either AB for assays with unregulated RhPh F-actin or was AB plus 50250 nM each Tn and Tm for regulated filaments. The concentrations of Tn and Tm in the wash buffer and motility buffer (see below) were the same and were chosen as the minimum needed to maintain regulation of the filaments, i.e., little or no movement at pCa 9 as previously described (20). A difference from our previous methods (20) was that regulated filaments were reconstituted on the flow cell surface by incubating unregulated RhPh-labeled F-actin filaments with AB plus 50250 nM each of Tn and Tm for 3 min, similar to our recent work with filaments regulated with sTn plus Tm (36).
Last, ATP-containing motility buffer was infused into the flow cell. Motility buffer for regulated actin filaments consisted of 2 mM Mg-ATP, 1 mM Mg2+, 65 mM Na+ + K+, 10 mM EGTA, 829 mM propionate, 2870 mM 3-(N-morpholino)propanesulfonic acid (MOPS), 0.085 M ionic strength (
/2), and 0.50.75% (wt/vol) methylcellulose, pH 7.0 at 30°C, the experimental temperature (20). Calcium dipropionate was altered to change the pCa (-log[Ca2+]) between 9.2 and 4.6 as calculated using the National Institute of Standards and Technology (NIST) Critically Selected Stability Constants of Metal Complexes Database. Motility buffer for assays of regulated actin contained 50250 nM each of Tm and Tn. For all motility buffers, 3 mg/ml glucose, 100 µg/ml glucose oxidase (Sigma, St. Louis, MO), 18 µg/ml catalase (Boehringer-Mannheim, Indianapolis, IN), and 40 mM DTT (Bio-Rad, Hercules, CA) were added to minimize photo-oxidation and photobleaching (34). Motility assays at 30°C were imaged and recorded on videocassettes as previously described (20).
Motion analysis.
Edge-detection hardware and Expert Vision software from Motion Analysis Systems (Santa Rosa, CA) were used to obtain filament speed statistics from videocassette recordings (20, 25, 59). Typically, six fields were analyzed for 1 min each in every flow cell. Data were sampled at 10 frames per second (fps) and individual filament paths, calculated from filament centroids, were retained only when they could be unambiguously tracked for at least 2 s. Speed statistics were calculated for each retained path using the Motion Analysis algorithm. The ratio of standard deviation (SD) to mean speed was calculated for each path as an indicator of uniformity of motion (13, 20, 25, 56, 59). A filament was accepted as moving uniformly when this ratio was <0.5 for 10 fps sampling. For each flow cell (one condition), the fraction of filaments moving uniformly and the unweighted mean speed (±SD) of those uniformly moving filaments (su) was obtained by combining information from all filament paths. If su was < 5 µm/s, then the position data along each path were smoothed with an unweighted moving average filter (5-frame window) and every fifth point retained, yielding an effective sampling rate of 2 fps; a filament was accepted as moving uniformly when the ratio of SD to mean speed was <0.3 for 2 fps sampling. Speed-pCa relations were fit to Eq. 1 by nonlinear least squares regression (SigmaPlot; SPSS, Richmond, CA):
 | (1) |
where smax is the speed at high [Ca2+] (low pCa), smin is the speed at low [Ca2+] (high pCa), pCa50 is an indicator of Ca2+ sensitivity and is the pCa needed to achieve 50% of smax, and n reflects the steepness of the relation and is typically used as an indicator of cooperativity.
Cardiac Trabecular Mechanics
Trabecular dissection and mechanical apparatus.
Mechanical experiments were conducted on permeabilized trabeculae from the right ventricular free walls of rat hearts obtained as described previously (22, 57). Adult male Sprague-Dawley rats were euthanized with pentobarbital (50 mg/kg ip). Hearts were rapidly excised and rinsed of blood, and the right ventricle splayed open in oxygenated physiological saline (in mM, 94 NaCl, 24 NaCO3, 5 KCl, 1 MgSO4, 1 Na2HPO4, 0.7 CaCl2) on a chilled dissection stage. The free wall was pinned out and incubated, with one change of solution, overnight at 4°C in skinning buffer (in mM: 100 KCl, 10 MOPS, 5 dipotassium EGTA, 9 MgCl2, 4 ATP, pH 7.0 at 4°C, 1% vol/vol Triton X-100, and 50% vol/vol glycerol). Solution was then changed to skinning buffer without Triton X-100 glycerol for dissection and storage. Individual trabeculae were dissected, and the ends were wrapped in photochemically etched T-clips and stored at -20°C for up to 4 days. Trabeculae were attached via T-clips to a force transducer (model 400A, 2.2-kHz resonant frequency; Cambridge Technology, Watertown, MA) at one end and a servo-motor (model 300; Cambridge Technology) tuned for a 300-µs step response at the opposite end; the mechanical apparatus was mounted on the stage of an inverted microscope (Leitz Diavert, Wetzlar, Germany) equipped for digital imaging (XR-77 CCD; Sony).
Solutions.
Solutions for experiments on permeabilized trabeculae contained (in mM) 15 phosphocreatine (PCr), 15 EGTA, at least 40 MOPS, 1 free Mg2+, 135 Na+ + K+, 1 DTT, 250 U/ml creatine kinase (CK; Sigma), and 5 ATP at pH 7.0 and 15 ± 1°C, the temperature at which mechanical measurements were made. Ionic strength was 0.2 M. For activation solutions, the Ca2+ level (expressed as pCa = -log[Ca2+]) was set to pCa 4.5 or pCa 4.0 by adjusting calcium dipropionate (propionate was the anion used to adjust ionic strength) (40, 57). Solutions were contained in 200-µl wells mounted on a temperature-controlled base. The base holds a total of 12 solution wells. The temperature of groups of four wells could be independently maintained by Peltier thermoelectric chips controlled by an ATR-4 adaptable thermoregulator (Quest Scientific, North Vancouver, BC, Canada) to aid in the cTn exchange protocol.
Isometric force.
Relaxed trabecular sarcomere length (Ls) was set to 2.2 µm with helium-neon laser diffraction (11). Trabecular length was 1.49 ± 0.35 mm and diameter was 147 ± 59 µm (mean ± SD, n = 23). Steady-state isometric force measurements were obtained under relaxing and activating conditions using our previously described data acquisition and control system (11, 12, 57). Trabecular length was shortened (by
20%) and rapidly restretched at 5-s intervals to maintain structural and mechanical integrity of the preparation (6, 10, 65); isometric force measurements were made during the steady-state period between shortening ramps, and the passive force (pCa 9) was subtracted to obtain active force (pCa
6). Force measurements (F) were then fit by nonlinear least squares regression (SigmaPlot; SPSS) to the Hill equation (Eq. 2)
 | (2) |
where Fmax is the force at high [Ca2+] (low pCa) and, as in Eq. 1, pCa50 is an indicator of Ca2+ sensitivity (pCa needed to achieve 50% of Fmax), and n reflects the steepness of the relation.
Exchange of recombinant cTn into permeabilized trabeculae.
To exchange purified troponins for endogenous troponin in permeabilized cardiac trabeculae, we adapted the method of Brenner et al. (8) devised for rabbit skeletal muscle. After initial control force data had been obtained, the trabecula was transferred first briefly (<1 min) to "pre-rigor" solution (in mM: 10 imidazole, 2.5 EGTA, and 15 EDTA) at 5°C. The fiber was then transferred into rigor solution [relaxing solution with no ATP, no PCr, no CK, and 5 mM 2,3-butanedione monoxime (BDM) added]. BDM was added to inhibit actomyosin force generation (1, 18), and the trabecula was also shortened to further eliminate rigor force. After 30 min in rigor solution, the trabecula was transferred into troponin exchange buffer (in mM: 20 MOPS, 5 MgCl2, 5 EGTA, 240 KCl, 5 DTT, 5 BDM, and 0.02 pepstatin, pH 6.5, plus
1 mg/ml cTn) at 10°C and covered to minimize evaporation or condensation. Typically, the trabecula was incubated in troponin exchange buffer for 120 min to achieve complete exchange (see RESULTS). The incubation time was abbreviated in some experiments to evaluate extent of exchange. At the end of the cTn exchange period, the trabecula was returned to pCa 9 relaxing solution at 15°C, and Ls was reset to 2.2 µm prior to data acquisition.
Fluorescence microscopy and laser-scanning confocal microscopy of trabeculae.
Fluorescence of trabeculae exchanged with cTn (5'-IATR-cTnT) was obtained as previously described for cardiac and skeletal muscle preparations following TnC replacement with 5'-IATR-labeled TnC (41, 42). To avoid nonspecific binding of fluorescently labeled cTn, the exchange protocol was modified by incubating trabeculae with 1 mg/ml BSA (in relaxing solution) for 10 min. Trabeculae that were to be examined by laser-scanning confocal microscopy were chemically fixed at the end of the experiment. For double-labeling experiments, trabeculae were incubated with phalloidin green (Molecular Probes, Eugene, OR) in relaxing solution prior to fixation. Trabeculae were dunked into two washes of rigor solution before being placed in 25 mM glutaraldehyde (in rigor) for 10 min. Confocal images were acquired with a Bio-Rad MRC-600 laser-scanning confocal microscope.
 |
RESULTS
|
---|
Unloaded Sliding of Single Actin Filaments
To test the effects of mutant cTnIs on actomyosin function, we first investigated sliding of isolated, well-regulated RhPh-actin filaments in an in vitro motility assay. Figure 1 shows the Ca2+ dependence of filament sliding speed over a surface coated with HMM from rabbit skeletal muscle. Each point in Fig. 1 represents data from one flow cell and is the average speed ± SD of 2242,419 uniformly moving filament paths that could be unambiguously tracked for at least 2 s (METHODS). Assays conducted with either WT cTn or with cTn reconstituted from WT cTnC, WT cTnT, and cTnI,K206Q exhibit minimal motility at low [Ca2+] (high pCa) and a graded increase of speed with decreasing pCa until a maximum was attained at high [Ca2+] (low pCa). Two effects of the K206Q mutation are evident in the data: 50% enhancement of the maximum sliding speed (smax) at high [Ca2+] and increased Ca2+ sensitivity (0.4 pCa unit leftward shift of the speed-pCa relation).

View larger version (36K):
[in this window]
[in a new window]
|
Fig. 1. Marked enhancement of maximum sliding speed and Ca2+ sensitivity of filament sliding of single, regulated actin filaments by cTnI,K206Q. In vitro motility assays were conducted with rabbit skeletal heavy meromyosin (HMM), actin, and Tm with wild-type (WT) cardiac troponin (cTn; open circles) or cTn reconstituted from WT cTnC, WT cTnT, and cTnI,K206Q (solid squares). Note that recombinant cTn subunit genes were from rat, but residue numbering refers to the human sequence to avoid confusion with the clinical literature (see METHODS). Points are average ± SD speed from one flow cell (n = 2242,419 filament paths per flow cell, which corresponds to a total from all 58 flow cells of >3.8 x 106 frame-to-frame determinations of speed). Lines were drawn according to the nonlinear least squares regressions according to Eq. 1. Regression parameter estimates of pCa50 were 5.84 ± 0.27 for WT (R2 = 0.830) and 6.23 ± 0.05 for cTnI,K206Q (R2 = 0.956) (see Fig. 2). Inset: video image of regulated, rhodamine-phalloidin (RhPh)-labeled actin filaments at pCa 5.
|
|
In vitro motility results similar to those shown in Fig. 1 were obtained with regulated actin containing either cTnI,K183
or cTnI,G203S (Fig. 2). Filament sliding was halted at low [Ca2+] for all regulated filaments containing either WT or mutant cTnI (Fig. 2A). The concentration of mutant cTns required to stop motility at pCa 9 was less than or equal to that for WT cTn, suggesting that the mutations do not reduce affinity of cTn for actin-Tm filaments. The maximum speed at high [Ca2+] (obtained from nonlinear regression of the data using Eq. 1) for all three mutants was elevated 4761% above that for WT (Fig. 2A) and also above the average sliding speed for unregulated F-actin, which does not vary with [Ca2+] (20). Ca2+ sensitivity, as indicated by pCa50, was altered with all three mutants such that less Ca2+ was required to activate filament sliding than with WT cTn (Fig. 2B).

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 2. In vitro motility speed-pCa regression parameter estimates in the presence of 50250 nM Tm and cTn containing either WT cTnI, cTnI,K183 , cTnI,G203S, or cTnI,K206Q. Average speed (±SD) of unregulated actin is shown for comparison in A ("unreg"). Nonlinear least squares regression was used to fit in vitro motility data to Eq. 1, as illustrated in Fig. 1. A: minimum speed (low [Ca2+]; open bars), maximum speed (high [Ca2+]; hatched bars), and (B) pCa50 are shown for regulated actin filaments with error bars representing SE of regression. Maximum speed for mutants was significantly higher than for WT (**P < 0.01). Maximum speed for WT was not significantly different from unregulated (P > 0.05). There were no significant differences between minimum speeds for regulated actin (P > 0.05). All assays were conducted with rabbit skeletal HMM.
|
|
It is possible that cTnI mutations could affect regulated motility assays differently when HMM from
- or ß-cardiac myosin is the motor protein rather than the faster rabbit skeletal HMM used in the initial experiments (Figs. 1 and 2). To test this possibility, we measured the maximum sliding speed obtained at low pCa (high [Ca2+]) using HMM purified from control rat hearts [predominantly
-myosin heavy chain (
-MHC)] or from hearts from PTU-treated rats (predominantly ß-MHC). The maximum speed for regulated filaments was normalized to that obtained with WT cTn for each of the HMM types: 4.14 ± 0.61 µm/s (n = 8) with rabbit skeletal HMM, 1.40 ± 0.51 µm/s (n = 5) with untreated rat cardiac HMM, and 1.02 ± 0.03 µm/s (n = 2) with PTU-treated rat cardiac HMM. The high [Ca2+] data with rabbit skeletal HMM from Fig. 2A was replotted in Fig. 3A after normalization to WT. Figure 3, B and C, illustrates that a similar pattern was obtained with both
- and ß-cardiac MHC, although the small sample size with PTU-treated rat cardiac HMM (Fig. 3C) reduced the level of statistical significance. The maximum speed for regulated filaments containing mutant cTnI (K183
, G203S, or K206Q) cTn was increased over WT cTn with either skeletal or cardiac HMM by 1.3- to 2.4-fold.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 3. Maximum speed of in vitro motility with rabbit skeletal HMM (A), rat cardiac HMM (predominantly -MHC isoform) (B), and rat cardiac HMM prepared from propylthiouracil (PTU)-treated animals (predominantly ß-MHC isoform) (C). Assays with regulated actin contained Tm and either WT cTnI, cTnI,K183 , cTnI,G203S, or cTnI,K206Q. Data within each panel were normalized to speed with WT cTnI. Error bars indicate SD for normalized data except for WT error bars that illustrate proportional error (SD) of averaged data before normalization. Level of statistical significance is indicated for comparison with WT cTn and the same myosin (*P < 0.05; **P < 0.01). With WT cTn, speed was 4.14 ± 0.61 µm/s (n = 8) with rabbit skeletal HMM, 1.40 ± 0.51 µm/s (n = 5) with rat cardiac HMM, and 1.02 ± 0.03 µm/s (n = 2) with PTU-rat cardiac HMM.
|
|
Force Generation in Cardiac Muscle Preparations
To examine the physiological consequences of FHC-related mutations in cTnI in the intact sarcomere, we developed a method for exchanging troponin complex into permeabilized trabeculae from rat heart. This technique is based on the method of Brenner and colleagues (8) for replacement of endogenous troponin in rabbit skeletal muscle with sTn containing fluorescently labeled sTnI. To validate this technique in cardiac muscle, our initial experiments utilized troponins containing fluorescently labeled subunits.
Incorporation of cTn(5'-IATR-cTnT).
We first followed the time course of incorporation of cTn composed of recombinant rat WT cTnC and cTnI with purified bovine cTnT labeled with 5'-IATR (Fig. 4). We chose to work initially with cTn containing fluorescently labeled cTnT because of the central role of TnT for anchoring the troponin complex to Tm (21, 51); incorporation of fluorescent cTnT into the sarcomere shows that the entire troponin complex has been incorporated. Bovine cTnT was used because it contains a Cys residue 39, whereas rat cTnT does not. The time course of cTn incorporation, as measured by monitoring total fluorescence over time, is shown in Fig. 4A. Fluorescence increased over the first 90 min, then decreased at 120 min, must likely due to slight photobleaching of the fluorescent label during repeated measurements. Maximum Ca2+-activated force was lower than the initial control in these preparations (27 ± 16%, n = 3), suggesting that the fluorescent probe (or possibly the substitution of bovine for rat cTnT) interfered with activation.

View larger version (67K):
[in this window]
[in a new window]
|
Fig. 4. Incorporation of exogenous troponin into permeabilized cardiac trabeculae. For the experiments shown here, troponin complex was formed with recombinant rat WT cTnC and WT cTnI and 5'-IATR-labeled cTnT purified from bovine heart. A: time course of cTn incorporation. Protocol for cTn exchange was interrupted at 30-min intervals to measure fluorescence intensity. Total fluorescence intensity was normalized to that obtained at 2 h. Points are single determinations, and line is nonlinear least squares regression fit to y = 1 - e-t/ . BF: confocal microscope images of cardiac trabeculae incubated for 15 min (B and C), 90 min (D and E), or 120 min (F) with 5'-IATR-labeled cTn. Scale bars represent 10 µm (B and D) and 2 µm (C, E, and F). Insets in B and D show florescence intensity profiles across diameter of the trabecular preparations.
|
|
Localization of cTn(5'-IATR-cTnT) was examined by confocal microscopy. At 15 min, the fluorescent label was unevenly distributed across the diameter of the trabecula (Fig. 4B) and along thin filaments (Fig. 4C). A radial gradient of fluorescence is consistent with diffusional transport of cTn into the myofilament lattice (Fig. 4B) with a lower limit of the diffusion coefficient D
2.4 x 10-9 cm2/s. At high magnification, fluorescent bands at the periphery of the trabecula were significantly shorter than the 2 µm expected for complete labeling of the I-band (Fig. 4C). This incomplete labeling at short times is consistent with Tn in the overlap zone being preferentially replaced first (60). In contrast, at 90 min, there was no obvious radial gradient of fluorescence across the trabecula (Fig. 4D), although there are presumptive interstitial spaces that are not labeled. I-bands were fully labeled along their entire length after 90 min of incubation (Fig. 4E). The latter point is particularly evident in a highly stretched region of another preparation labeled for 120 min (Fig. 4F). Z-lines are evident as thin dark stripes within the fluorescent bands of Fig. 4, E and F. This pattern suggests that cTn binding is specific to thin filaments and that nonspecific binding is minimal. We therefore chose 2 h as the routine time for incubation with cTn, similar to that used in the protocol for skeletal muscle (8), to ensure full incorporation of the desired protein.
Recombinant rat WT cTn.
After demonstrating incorporation of endogenous cTn into trabecular preparations, we measured steady-state isometric force-pCa relations before and after exchange with WT cTn (Fig. 5A). These data were acquired to control for effects of the exchange procedure and serve as the baseline against which cTn containing mutant subunits is compared (each preparation, prior to exchange also serves as its own control). WT cTn was prepared from recombinant WT cTnC, WT cTnI, and WT cTnT with rat sequences (METHODS) and thus was similar to the endogenous complex except for presence of NH2-terminal Met residues combined with the absence of an acetylated NH2 terminus, absence of phosphorylated residues, and presence of only a single, adult isoform of cTnT. Figure 5B shows that trabecular structure and striation pattern were well maintained throughout the entire experimental protocol. Following the exchange procedure, Fmax (pCa 4) was 86 ± 10% (mean ± SD) of the initial control (Fig. 5C, inset). Before exchange, pCa50 (Eq. 2) was 5.37 ± 0.10 (mean ± SD, n = 10) and was 5.33 ± 0.09 after WT cTn exchange (Fig. 5C). Hill coefficient n was 6.6 ± 1.5 before and 4.7 ± 1.8 after exchange. Comparable results were obtained in five preparations using native cTn purified from rat heart, suggesting that changes were not due to differences between recombinant and native proteins (Fig. 5C). Overall, these control measurements demonstrate that the procedure for cTn exchange in cardiac trabeculae causes only small changes in Ca2+-activated, steady-state force.

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 5. Incorporation of recombinant WT cTn into permeabilized rat cardiac trabeculae. A: slow time-base recording of force. Activation pCa is indicated below record; relaxing pCa 9 otherwise. Transients in the force record occur at 5-s intervals due to periodic shortening/restretch that briefly unloads the muscle preparation (see METHODS). In the first half of the record, control force-pCa data were acquired prior to cTn exchange. Breaks in the force record are associated with prolonged incubation with recombinant WT cTn. B: digital image of a rat cardiac trabecula at the end of a troponin exchange experiment (pCa 9). Diameter = 90 µm. Sarcomere length = 2.2 µm. Note preservation of sarcomere structure. C: summary of steady-state, isometric force-pCa relations from cardiac trabeculae prior to (solid circles) and following incubation with WT cTn (open circles). Data were normalized to force obtained at pCa 4. Points are means ± SE (n = 10 trabeculae). Lines were drawn according to Eq. 2 using average regression parameter estimates. Average regression parameter estimates of pCa50 were 5.37 ± 0.03 before and 5.33 ± 0.03 after exchange. Average estimates of n were 6.59 ± 0.49 before and 4.66 ± 0.57 after exchange. Inset: small reduction in maximum isometric force to 86% of initial control at pCa 4.
|
|
Recombinant cTn(Rh-cTnC,D65A).
After demonstrating that exogenous cTn can be incorporated into permeabilized trabeculae (Fig. 4) with minimal effects on steady-state force (Fig. 5), it was necessary to further verify that exogenous cTn functionally replaces endogenous cTn and to estimate the extent of replacement. To accomplish this, we reconstituted cTn from WT cTnT and WT cTnI with rhodamine-labeled cTnC,D65A. The D65A mutation eliminates Ca2+ binding to site II, the sole Ca2+ trigger site of cTnC, at physiologically relevant [Ca2+]. We refer to this mutant protein as xcTnC to parallel our recent studies of the analogous double mutant (mutations at sites I and II) of sTnC, xxsTnC (58). Rat xcTnC is comparable to mutant CBMII ("calcium binding mutant at site II") used by others (28, 45, 53). Steady-state force measurements at pCa 4 show that only
2% of the initial control force is present after exchange with cTn containing Rh-xcTnC (Fig. 6A) demonstrating that exogenous cTn functionally replaces almost all of the endogenous cTn during the exchange procedure. Localization of the exogenous cTn to actin filaments was verified by confocal microscopy (Fig. 6, BD). Colocalization of Rh-labeled cTn (Fig. 6C) and phalloidin-green-labeled actin (Fig. 6B) is clearly evident in the average sarcomere scans of Fig. 6, B and C (Fig. 6D).

View larger version (56K):
[in this window]
[in a new window]
|
Fig. 6. Functional validation of exogenous troponin exchange into cardiac trabeculae. For the experiments shown here, troponin complex was formed with recombinant rat WT cTnI, WT cTnT, and Rh-labeled cTnC,D65A (Rh-xcTnC), a mutation that renders cTn unable to activate contraction. A: force record illustrating a cardiac trabeculae which produced 55 mN/mm2 of active force (pCa 4, shown below force record) prior to cTn exchange but only 2% of the original value after exchange with cTn containing the mutant xcTnC (break in record), indicating that essentially all endogenous had been replaced by the exogenous protein. B and C: confocal microscope images showing colocalization of phalloidin green (actin) (B) and Rh-xcTnC (C). D: fluorescence intensity scans of phalloidin green (green) and Rh-xcTnC (red) images. Fluorescence intensity was averaged over the region delimited by white, dotted lines in C and the equivalent region in B. Fluorescence intensity is in arbitrary units, and the phalloidin green scan was offset relative to the Rh-xcTnC scan for clarity. Length axis for D applies to BD.
|
|
Recombinant cTn(cTnI,K206Q).
To determine the effects of FHC-related mutations on isometric force, we utilized the cTn exchange procedure with cTn containing mutant cTnI,K206Q. The K206Q mutation was chosen because results in motility assays were representative of all three mutations studied (Figs. 2 and 3). Fmax (pCa 4) was 84 ± 24% (mean ± SD) of the initial control following the exchange procedure (Fig. 7, inset). Before exchange, pCa50 (Eq. 2) was 5.38 ± 0.02 (mean ± SD, n = 8) and was 5.34 ± 0.04 after cTn(cTnI,K206Q) exchange (Fig. 7). Hill coefficient n was 7.7 ± 3.6 before and 3.9 ± 0.5 after exchange. There were no significant differences between these parameters obtained with cardiac trabeculae containing cTn(cTnI,K206Q) compared with those from trabeculae containing WT cTn (Fig. 7), indicating that this mutation has little or no effect on Ca2+ activation of steady-state force development under isometric conditions.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 7. Lack of effect of cTnI,K206Q mutation on Ca2+ dependence of steady-state isometric force. Force-pCa relations from cardiac trabeculae following incubation with WT cTn (open circles, replotted from Fig. 5C) or cTn reconstituted from WT cTnC, WT cTnT, and cTnI,K206Q (solid squares). Data were normalized to force obtained at pCa 4. Points are means ± SE (n = 8 trabeculae with cTnI,K206Q). Lines were drawn according to Eq. 2 using average regression parameter estimates. Average regression parameter estimates of pCa50 were 5.33 ± 0.03 for WT exchange and 5.34 ± 0.01 for cTnI,K206Q exchange. Average estimates of n were 4.66 ± 0.57 after WT exchange and 3.92 ± 0.17 after cTnI,K206Q exchange. Inset: reduction in maximum isometric force (pCa 4) after exchange to 86% of the pre-exchange value for WT and to 84% for cTnI,K206Q.
|
|
 |
DISCUSSION
|
---|
In this study, we found the following. 1) Compared with WT cTnI, mutations K183
, G203S. and K206Q substantially enhanced Ca2+ sensitivity and, surprisingly, maximum speed of filament sliding in the in vitro motility assay using regulated F-actin containing recombinant subunits of rat cTn. 2) Maximum sliding speed was enhanced irrespective of whether HMM was derived from rabbit skeletal muscle, control rat cardiac muscle (primarily
-MHC), or cardiac muscle from PTU-treated rats (primarily ß-MHC). 3) At very low [Ca2+], there was no change in effectiveness of the mutant cTnIs examined to inhibit filament sliding in the in vitro motility assay and force generation of permeabilized trabeculae. 4) Exogenous cTn can be efficiently exchanged for endogenous cTn in permeabilized trabeculae from cardiac muscle. 5) cTnI mutation K206Q did not affect the Ca2+ sensitivity or Fmax of permeabilized trabecular preparations. Taken together, these results suggest an important functional role for the COOH terminus of TnI in determining the duration of actomyosin transitions that limit filament sliding speed, but not isometric force generation, and implicate enhanced contractility in the beating heart as the initial signal in the cTnI-related pathway(s) to hypertrophy.
Procedure for cTn exchange into permeabilized cardiac preparations.
One significant aspect of this study is methodological. We have adapted a method for Tn complex exchange established for skeletal muscle (8) to permeabilized cardiac muscle. This novel method is a preferable alternative to methods using troponin subunits, particularly TnT (23, 24, 61) or orthovanadate (64). Both of these other methods lead to significant active force in the absence of Ca2+ during the exchange procedure, and the latter method does not allow replacement of cTnT. Thus the method reported here will be generally useful for introduction of mutant proteins and proteins modified with a variety of labels (fluorescent, etc.) into cardiac preparations.
Figures 46 show that exogenous cTn can be stably incorporated into the correct, functional binding sites on actin filaments of intact cardiac sarcomeres over a time course of 2 h, with minimal change in function or trabecula structure due to the exchange procedure itself or to the minor differences between recombinant troponin subunits and the endogenous proteins. The small decrease in Ca2+ sensitivity observed after exchange with WT cTn (Fig. 5) could not be explained by the absence of phosphorylation of recombinant cTnI. Replacing endogenous cTnI with unphosphorylated, recombinant cTnI would be expected to have either no effect or to increase Ca2+ sensitivity (63), whereas we observed a small decrease in Ca2+ sensitivity (Fig. 5). The changes in Ca2+ sensitivity and force related to the procedure may have resulted from a small loss of TnC (5, 46), although the more likely explanation is a general loss of function during the course of experiments with permeabilized preparations. The key modifications that minimize such loss of function involve reducing rigor force in the exchange buffer (METHODS).
Changes in myofilament function due to cTnI mutations.
The three COOH-terminal mutations in rat cTnI studied in the motility assay (K183
, G203S, and K206Q) all caused substantial increases in the Ca2+ sensitivity of filament sliding (Figs. 1 and 2B), a result that is in accord with observed enhancements of Ca2+ sensitivity of solution ATPase activity by the R145G, R145Q, R162W, K183
, and K206Q mutants (19, 67) but not with reports of the G203S mutation having no effect on ATPase (9, 67). A reduction in the effectiveness of TnI inhibition (i.e., less Ca2+ is required to turn on the filament) under unloaded conditions (filament sliding or acto-S1-ATPase activity in solution) is consonant with the studies of COOH-terminal truncation mutants of cTnI by Rarick et al. (54) and of skeletal TnI by Van Eyk et al. (71). Figures 1, 2A, and 7 all illustrate that the single residue mutations in this study were all effective at turning off actomyosin in the absence of Ca2+, also indicating complete reconstitution of regulated thin filaments, which differs from the effects observed in TnI truncation studies. Results with K183
, G203S, and K206Q mutants also differ from those with R145G that suggested the possibility of diastolic dysfunction for that specific mutation (9, 19, 35, 70).
The most dramatic and surprising effect of cTnI mutants K183
, G203S, and K206Q introduced into recombinant rat cTn was the large enhancement of smax (Figs. 1, 2A, and 3). Intriguingly, the enhancement was not only above smax for WT Tn but was also above the speed of unregulated F-actin alone (Fig. 2A). The speed of unregulated actin is similar to smax for filaments regulated with rat WT cTn (Fig. 2A) (25), although only the speed of regulated F-actin varies with [Ca2+] (20, 25).
At first glance, it seems surprising that the large enhancements obtained in the motility assay with rat cTnI,K206Q (Figs. 13) were not also observed in force-pCa relations of trabeculae (Fig. 7). There is a related example of such disparate effects on isometric force vs. unloaded sliding in the literature. An FHC-related mutation in troponin T, cTnT,I79N, also has been shown to have different effects on maximum sliding speed and isometric force. At saturating [Ca2+], this cTnT mutation increases smax of single filaments in motility assays by 750% (26, 37) and also increases Vmax of single fibers by 70% (66). Under isometric conditions, Fmax of single filaments was decreased by 26% (26) and by 27% in single fibers (66). This example in which isometric force decreased differs from the cTnI,K206Q mutation, which did not affect isometric force even though both mutations increased unloaded sliding speed. Significantly, this example shows that differences in isometric vs. unloaded parameters observed in the intact sarcomere persist at the single filament level.
Enhancements of smax observed in this study were not paralleled by increases in maximum ATPase activity in previous studies of the K183
and K206Q mutations (67); although Takahashi-Yanaga et al. (67) observed a small increase in the maximum ATPase with the G203S mutation, Burton et al. (9) did not. In our experiments, this enhancement with mutant cTns was observed with HMM from both skeletal and cardiac muscle sources (Fig. 3) and thus is not an artifact of using fast skeletal myosin. The enhancement of smax by mutant rat cTns is reminiscent of the effect of sTn (20) and supports the suggestion that regulatory proteins can uniquely modulate the kinetics of interaction of myosin cross-bridges with actin (20, 26, 66); it highlights a role for charged residues, particularly basic residues that are mutated in FHC, with G203S being an exception, and thus electrostatic interactions in this process.
Mechanism of increased sliding speed without affecting isometric force.
The example of cTnT,I79N mutation described above suggests that disparity between effects on isometric force vs. unloaded sliding is not unique to mutations in cTnI. How can this apparent contradiction be explained? Isometric force is proportional to f/(f + g), where f is the apparent rate constant for the transition from weak (non-force generating) states to strong (force generating) states, and g is the apparent rate constant for the return of cross-bridges to weak-binding states (7). The latter rate (g) is limited by ADP release. Under isometric conditions when cross-bridges are strained, g is slow (g1x/h in the framework of Ref. 27, where x is the displacement between a cross-bridges equilibrium position and the binding site on actin, and h is the maximum work-producing displacement of a cross-bridge); under these conditions, g is typically smaller than f and thus does not have a large effect on Fmax. On the other hand, if we assume that sliding speed in motility assays is proportional to the maximum shortening speed, Vmax, of the intact sarcomere, then filament sliding would be proportional to g2 (again in the framework of Ref. 27). Detachment rate g2 applies to force generating cross-bridges after strain is released during movement. The two detachment rates for isometric and unloaded conditions, g1x/h and g2, respectively, are not the same, and evidence points to g2 in unloaded conditions being substantially faster than g1x/h (27). To summarize, the step in the cross-bridge cycle most likely to increase filament sliding speed without influencing isometric tension, would be an increase in detachment rate g2 (but not g1) (27), and such a modulation of the cross-bridge cycle during unloaded filament sliding is a previously unappreciated function of the COOH terminus of cTnI.
The combined enhancements of smax and pCa50 for filament sliding imply that systolic pumping activity would be markedly enhanced throughout the entire Ca2+ transient. The data in Fig. 7 suggest that the amount of Ca2+ bound to the myofilaments, and by implication, free Ca2+ because TnC is a major site of myoplasmic Ca2+ binding, would not likely change, because Ca2+ affinity as reflected by Ca2+ sensitivity of isometric force was not significantly affected, at least for the cTnI (K206Q) mutation. [Ca2+ sensitivity of force is a better indicator of Tn affinity than filament sliding, because maximum speed for filament sliding is achieved with only partial activation of regulated thin filaments, as evidenced by motility speed-pCa relations (Fig. 1) being shifted leftward by
0.5 (WT) or
0.9 (K206Q) pCa units relative to comparable force-pCa curves (Fig. 7), in agreement with previous studies (20, 26, 36) that used other troponins]. If, as suggested above, g2 is increased by changes in the COOH terminus of TnI, then filament sliding speed would also increase at submaximal [Ca2+] without influencing isometric tension, because g2 is only relevant during filament sliding (27). Clearly, it is important to investigate both isometric conditions and more dynamic conditions, particularly filament sliding, because the heart muscle cells shorten during the ejection period of each beat. Thus the changes observed in the motility assay due to cTnI mutations are not in conflict with the lack of change in isometric force and are particularly relevant to the beating heart of affected individuals.
Mechanism of hypertrophy.
Initial studies of FHC-related mutations in myosin suggested that hypertrophy was the result of a compensatory response to reduced contractility (4, 48, 55). Subsequent studies, particularly those on thin filament Ca2+-regulatory protein mutations cTnT,I79N, cTnT,R92Q,
-Tm, D175N,
-Tm,E180G, and the cTnI mutations examined in this study, illustrate that hypertrophy could result from enhanced contractility as evidenced by increased filament sliding speed at saturating [Ca2+] (Figs. 13 and Refs. 2, 26, 37, 66). Furthermore, the majority of FHC-related mutations in cTnT (32), cTnI (Figs. 1 and 2; and Refs. 9, 19, 35, 67), and
-Tm (3, 30) lead to enhanced Ca2+ sensitivity of force and/or filament sliding, thus leading to enhanced cardiac function at [Ca2+] that is physiologically relevant to the cardiac contractile cycle. Such an effect could be mediated directly through elevated ATPase activity, or it could result from kinetically mediated alterations in cooperativity in the thin filament.
The possibility that there could be two or more pathways to muscular hypertrophy is plausible given the wide variety of stimuli that can cause a hypertrophic response in vitro and in vivo (68). Interestingly, recent examinations of MHC mutants R403Q and L908V suggest the possibility of convergence in the mechanisms underlying these seemingly different routes to hypertrophy. Tyska et al. (69) reported that MHC,R403Q and Palmiter et al. (49) reported that both MHC,R403Q and MHC,L908V mutations increase filament sliding speed of unregulated F-actin. MHC,R719W has also been reported to increase isometric force and stiffness in the intact sarcomere (33). These observations have not been reconciled with earlier reports that FHC-related mutations in MHC result in dominant-negative, inhibitory effects on contractility. They support the likelihood that mutations in ß-MHC cause hypertrophy through enhanced activity as is reported here for cTnI mutations. It remains to be determined whether multiple signaling pathways are involved in FHC, or whether there is a common underlying route to hypertrophy for the mutations identified in a wide variety of sarcomeric proteins.
 |
DISCLOSURES
|
---|
Funding was provided by National Institutes of Health Grants HL-63974, HL-52558, and NS-08384, by the American Heart Association-Washington State Affiliate, the University of Washington Royalty Research Fund, the Biomedical Science Exchange Program, and the Hannover Medical School. M. Regnier is and Established Investigator of the American Heart Association.
 |
ACKNOWLEDGMENTS
|
---|
We thank Paulette Brunner, Jennifer Fredlund, Josh Hawkins, Zhaoxiong Luo, Martha Mathiason, Robin Mondares, and Scott Myrick for excellent technical assistance; Dr. John E. T. Corrie for kindly supplying the 5' isomer of IATR; and Drs. Larry S. Tobacman and Earl Homsher for generous assistance in preliminary experiments.
Present address for Y. Chen: UMDNJ RWJ Medical School, Dept. Pathology, Piscataway, NJ 08854.
 |
FOOTNOTES
|
---|
1 This article was submitted in conjunction with the meeting entitled "Physiological Genomics of Cardiovascular Disease: from Technology to Physiology," in San Francisco, CA, February 2002, sponsored by the American Physiological Society.
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).
Address for reprint requests and other correspondence: P. B. Chase, Florida State Univ., Dept. of Biological Science and Program in Molecular Biophysics, Biology Unit One, Tallahassee, FL 32306-4370 (E-mail: chase{at}bio.fsu.edu).
10.1152/physiolgenomics.00101.2002. 
 |
References
|
---|
- Backx PH, Gao WD, Azan-Backx MD, and Marban E. Mechanism of force inhibition by 2,3-butanedione monoxime in rat cardiac muscle: roles of [Ca2+]i and cross-bridge kinetics. J Physiol 476: 487500, 1994.[Abstract]
- Bing W, Redwood CS, Purcell IF, Esposito G, Watkins H, and Marston SB. Effects of two hypertrophic cardiomyopathy mutations in
-tropomyosin, Asp175Asn and Glu180Gly, on Ca2+ regulation of thin filament motility. Biochem Biophys Res Commun 236: 760764, 1997.[ISI][Medline]
- Bing W, Knott A, Redwood C, Esposito G, Purcell I, Watkins H, and Marston S. Effect of hypertrophic cardiomyopathy mutations in human cardiac muscle
-tropomyosin (Asp175Asn and Glu180Gly) on the regulatory properties of human cardiac troponin determined by in vitro motility assay. J Mol Cell Cardiol 32: 14891498, 2000.[ISI][Medline]
- Bonne G, Carrier L, Richard P, Hainque B, and Schwartz K. Familial hypertrophic cardiomyopathy: from mutations to functional defects. Circ Res 83: 580593, 1998.[Abstract/Free Full Text]
- Brandt PW, Diamond MS, Rutchik JS, and Schachat FH. Co-operative interactions between troponin-tropomyosin units extend the length of the thin filament in skeletal muscle. J Mol Biol 195: 885896, 1987.[ISI][Medline]
- Brenner B. Technique for stabilizing the striation pattern in maximally calcium-activated skinned rabbit psoas fibers. Biophys J 41: 99102, 1983.[Abstract]
- Brenner B and Eisenberg E. Rate of force generation in muscle: correlation with actomyosin ATPase activity in solution. Proc Natl Acad Sci USA 83: 35423546, 1986.[Abstract]
- Brenner B, Kraft T, Yu LC, and Chalovich JM. Thin filament activation probed by fluorescence of N-((2-(iodoacetoxy) ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole-labeled troponin I incorporated into skinned fibers of rabbit psoas muscle. Biophys J 77: 26772691, 1999.[Abstract/Free Full Text]
- Burton D, Abdulrazzak H, Knott A, Elliott K, Redwood C, Watkins H, Marston S, and Ashley C. Two mutations in troponin I that cause hypertrophic cardiomyopathy have contrasting effects on cardiac muscle contractility. Biochem J 362: 443451, 2002.[ISI][Medline]
- Chase PB and Kushmerick MJ. Effects of pH on contraction of rabbit fast and slow skeletal muscle fibers. Biophys J 53: 935946, 1988.[Abstract]
- Chase PB, Martyn DA, Kushmerick MJ, and Gordon AM. Effects of inorganic phosphate analogues on stiffness and unloaded shortening of skinned muscle fibres from rabbit. J Physiol 460: 231246, 1993.[Abstract]
- Chase PB, Martyn DA, and Hannon JD. Activation dependence and kinetics of force and stiffness inhibition by aluminiofluoride, a slowly dissociating analogue of inorganic phosphate, in chemically skinned fibres from rabbit psoas muscle. J Muscle Res Cell Motil 15: 119129, 1994.[ISI][Medline]
- Chase PB, Chen Y, Kulin K, and Daniel TL. Viscosity and solute dependence of F-actin translocation by rabbit skeletal heavy meromyosin. Am J Physiol Cell Physiol 278: C1088C1098, 2000.[Abstract/Free Full Text]
- Chase PB, Köhler J, Chen Y, Wang CK, Luo Z, Kraft T, and Brenner B. Physiological effects of cardiac troponin I mutations related to familial hypertrophic cardiomyopathy. Biophys J 80: 342a343a, 2001.
- Chase PB. Proposed mechanism of hypertrophy due to C-terminal mutations in cardiac troponin I. Physiologist 45: 71, 2002.
- Chomczynski P and Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156159, 1987.[ISI][Medline]
- Dong W, Rosenfeld SS, Wang CK, Gordon AM, and Cheung HC. Kinetic studies of calcium binding to the regulatory site of troponin C from cardiac muscle. J Biol Chem 271: 688694, 1996.[Abstract/Free Full Text]
- Ebus JP and Stienen GJM. Effects of 2,3-butanedione monoxime on cross-bridge kinetics in rat cardiac muscle. Pflügers Arch 432: 921929, 1996.[ISI][Medline]
- Elliott K, Watkins H, and Redwood CS. Altered regulatory properties of human cardiac troponin I mutants that cause hypertrophic cardiomyopathy. J Biol Chem 275: 2206922074, 2000.[Abstract/Free Full Text]
- Gordon AM, LaMadrid M, Chen Y, Luo Z, and Chase PB. Calcium regulation of skeletal muscle thin filament motility in vitro. Biophys J 72: 12951307, 1997.[Abstract]
- Gordon AM, Homsher E, and Regnier M. Regulation of contraction in striated muscle. Physiol Rev 80: 853924, 2000.[Abstract/Free Full Text]
- Hancock WO, Martyn DA, Huntsman LL, and Gordon AM. Influence of Ca2+ on force redevelopment kinetics in skinned rat myocardium. Biophys J 70: 28192829, 1996.[Abstract]
- Hatakenaka M and Ohtsuki I. Replacement of three troponin components with cardiac troponin components within single glycerinated skeletal muscle fibers. Biochem Biophys Res Commun 181: 10221027, 1991.[ISI][Medline]
- Hatakenaka M and Ohtsuki I. Effect of removal and reconstitution of troponins C and I on the Ca2+-activated tension development of single glycerinated rabbit skeletal muscle fibers. Eur J Biochem 205: 985993, 1992.[Abstract]
- Homsher E, Kim B, Bobkova A, and Tobacman LS. Calcium regulation of thin filament movement in an in vitro motility assay. Biophys J 70: 18811892, 1996.[Abstract]
- Homsher E, Lee DM, Morris C, Pavlov D, and Tobacman LS. Regulation of force and unloaded sliding speed in single thin filaments: effects of regulatory proteins and calcium. J Physiol 524: 233243, 2000.[Abstract/Free Full Text]
- Huxley AF. Muscle structure and theories of contraction. Prog Biophys 7: 255318, 1957.[ISI]
- Huynh Q, Butters CA, Leiden JM, and Tobacman LS. Effects of cardiac thin filament Ca2+: statistical mechanical analysis of a troponin C site II mutant. Biophys J 70: 14471455, 1996.[Abstract]
- James J, Zhang Y, Osinska H, Sanbe A, Klevitsky R, Hewett TE, and Robbins J. Transgenic modeling of a cardiac troponin I mutation linked to familial hypertrophic cardiomyopathy. Circ Res 87: 805811, 2000.[Abstract/Free Full Text]
- Karibe A, Tobacman LS, Strand J, Butters C, Back N, Bachinski LL, Arai AE, Ortiz A, Roberts R, Homsher E, and Fananapazir L. Hypertrophic cardiomyopathy caused by a novel
-tropomyosin mutation (V95A) is associated with mild cardiac phenotype, abnormal calcium binding to troponin, abnormal myosin cycling, and poor prognosis. Circulation 103: 6571, 2001.[Abstract/Free Full Text]
- Kimura A, Harada H, Park JE, Nishi H, Satoh M, Takahashi M, Hiroi S, Sasaoka T, Ohbuchi N, Nakamura T, Koyanagi T, Hwang TH, Choo JA, Chung KS, Hasegawa A, Nagai R, Okazaki O, Nakamura H, Matsuzaki M, Sakamoto T, Toshima H, Koga Y, Imaizumi T, and Sasazuki T. Mutations in the cardiac troponin I gene associated with hypertrophic cardiomyopathy. Nat Genet 16: 379382, 1997.[ISI][Medline]
- Knollmann BC and Potter JD. Altered regulation of cardiac muscle contraction by troponin T mutations that cause familial hypertrophic cardiomyopathy. Trends Cardiovasc Med 11: 206212, 2001.[ISI][Medline]
- Köhler J, Winkler G, Schulte I, Scholz T, McKenna W, Brenner B, and Kraft T. Mutation of the myosin converter domain alters cross-bridge elasticity. Proc Natl Acad Sci USA 99: 35573562, 2002.[Abstract/Free Full Text]
- Kron SJ, Toyoshima YY, Uyeda TQP, and Spudich JA. Assays for actin sliding movement over myosin-coated surfaces. Methods Enzymol 196: 399416, 1991.[ISI][Medline]
- Lang R, Gomes AV, Zhao J, Housmans PR, Miller T, and Potter JD. Functional analysis of a troponin I (R145G) mutation associated with familial hypertrophic cardiomyopathy. J Biol Chem 277: 1167011678, 2002.[Abstract/Free Full Text]
- Liang B, Chen Y, Wang C-K, Luo Z, Regnier M, Gordon AM, and Chase PB. Ca2+ regulation of rabbit skeletal muscle thin filament sliding: role of cross-bridge number. Biophys J In press.
- Lin D, Bobkova A, Homsher E, and Tobacman LS. Altered cardiac troponin T in vitro function in the presence of a mutation implicated in familial hypertrophic cardiomyopathy. J Clin Invest 97: 28422848, 1996.[Abstract/Free Full Text]
- Malik MSA and Watkins H. The molecular genetics of hypertrophic cardiomyopathy. Curr Opin Cardiol 12: 295302, 1997.[ISI][Medline]
- Maron BJ. Hypertrophic cardiomyopathy. Lancet 350: 127133, 1997.[ISI][Medline]
- Martyn DA, Chase PB, Hannon JD, Huntsman LL, Kushmerick MJ, and Gordon AM. Unloaded shortening of skinned muscle fibers from rabbit activated with and without Ca2+. Biophys J 67: 19841993, 1994.[Abstract]
- Martyn DA, Freitag CJ, Chase PB, and Gordon AM. Ca2+ and cross-bridge-induced changes in troponin C in skinned skeletal muscle fibers: effects of force inhibition. Biophys J 76: 14801493, 1999.[Abstract/Free Full Text]
- Martyn DA, Regnier M, Xu D, and Gordon AM. Ca2+- and cross-bridge-dependent changes in N- and C-terminal structure of troponin C in rat cardiac muscle. Biophys J 80: 360370, 2001.[Abstract/Free Full Text]
- Mogensen J, Klausen IC, and Egeblad H. Sudden cardiac death in familial hypertrophic cardiomyopathy is associated with a novel mutation in the troponin I gene. Circulation 100: I-618, 1999.
- Mörner S, Richard P, Kazzam E, Hainque B, Schwartz K, and Waldenström A. Deletion in the cardiac troponin I gene in a family from northern Sweden with hypertrophic cardiomyopathy. J Mol Cell Cardiol 32: 521525, 2000.[ISI][Medline]
- Morris CA, Tobacman LS, and Homsher E. Modulation of contractile activation in skeletal muscle by a calcium-insensitive troponin C mutant. J Biol Chem 276: 2024520251, 2001.[Abstract/Free Full Text]
- Moss RL, Giulian GG, and Greaser ML. The effects of partial extraction of TnC upon the tension-pCa relationship in rabbit skinned skeletal muscle fibers. J Gen Physiol 86: 585600, 1985.[Abstract]
- Niimura H, Patton KK, McKenna WJ, Soults J, Maron BJ, Seidman JG, and Seidman CE. Sarcomere protein gene mutations in hypertrophic cardiomyopathy of the elderly. Circulation 105: 446451, 2002.[Abstract/Free Full Text]
- Palmiter KA and Solaro RJ. Molecular mechanisms regulating the myofilament response to Ca2+: implications of mutations causal for familial hypertrophic cardiomyopathy. Basic Res Cardiol 92: 6374, 1997.[ISI][Medline]
- Palmiter KA, Tyska MJ, Haeberle JR, Alpert NR, Fananapazir L, and Warshaw DM. R403Q and L908V mutant beta-cardiac myosin from patients with familial hypertrophic cardiomyopathy exhibit enhanced mechanical performance at the single molecule level. J Muscle Res Cell Motil 21: 609620, 2000.[ISI][Medline]
- Pardee JD and Spudich JA. Purification of muscle actin. Methods Enzymol 85: 164181, 1982.[ISI][Medline]
- Perry SV. Troponin T: genetics, properties and function. J Muscle Res Cell Motil 19: 575602, 1998.[ISI][Medline]
- Potter JD. Preparation of troponin and its subunits. Methods Enzymol 85: 241263, 1982.[ISI][Medline]
- Putkey JA, Sweeney HL, and Campbell ST. Site-directed mutation of the trigger calcium-binding sites in cardiac troponin C. J Biol Chem 264: 1237012378, 1989.[Abstract/Free Full Text]
- Rarick HM, Tu XH, Solaro RJ, and Martin AF. The C terminus of cardiac troponin I is essential for full inhibitory activity and Ca2+ sensitivity of rat myofibrils. J Biol Chem 272: 2688726892, 1997.[Abstract/Free Full Text]
- Redwood CS, Moolman-Smook JC, and Watkins H. Properties of mutant contractile proteins that cause hypertrophic cardiomyopathy. Cardiovasc Res 44: 2036, 1999.[ISI][Medline]
- Regnier M, Martyn DA, and Chase PB. Calmidazolium alters Ca2+ regulation of tension redevelopment rate in skinned skeletal muscle. Biophys J 71: 27862794, 1996.[Abstract]
- Regnier M, Rivera AJ, Chen Y, and Chase PB. 2-Deoxy ATP enhances contractility of rat cardiac muscle. Circ Res 86: 12111217, 2000.[Abstract/Free Full Text]
- Regnier M, Rivera AJ, Bates MA, Wang CK, Chase PB, and Gordon AM. Thin filament near-neighbor regulatory unit interactions affect rabbit skeletal muscle steady-state force-Ca2+ relations. J Physiol 540: 485497, 2002.[Abstract/Free Full Text]
- Sellers JR, Cuda G, Wang F, and Homsher E. Myosin-specific adaptations of the motility assay. Methods Cell Biol 39: 2349, 1993.[ISI][Medline]
- She M, Trimble D, Yu LC, and Chalovich JM. Factors contributing to troponin exchange in myofibrils and in solution. J Muscle Res Cell Motil 21: 737745, 2000.[ISI][Medline]
- Shiraishi F, Kambara M, and Ohtsuki I. Replacement of troponin components in myofibrils. J Biochem (Tokyo) 111: 6165, 1992.[Abstract]
- Smillie LB. Preparation and identification of
- and ß-tropomyosins. Methods Enzymol 85: 234241, 1982.[ISI][Medline]
- Solaro RJ and Rarick HM. Troponin and tropomyosin: proteins that switch on and tune in the activity of cardiac myofilaments. Circ Res 83: 471480, 1998.[Abstract/Free Full Text]
- Strauss JD, Zeugner C, Van Eyk JE, Bletz C, Troschka M, and Rüegg JC. Troponin I replacement in permeabilized cardiac muscle. Reversible extraction of troponin I by extraction with vanadate. FEBS Lett 310: 229234, 1992.[ISI][Medline]
- Sweeney HL, Corteselli SA, and Kushmerick MJ. Measurements on permeabilized skeletal muscle fibers during continuous activation. Am J Physiol Cell Physiol 252: C575C580, 1987.[Abstract/Free Full Text]
- Sweeney HL, Feng HS, Yang Z, and Watkins H. Functional analyses of troponin T mutations that cause hypertrophic cardiomyopathy: insights into disease pathogenesis and troponin function. Proc Natl Acad Sci USA 95: 1440614410, 1998.[Abstract/Free Full Text]
- Takahashi-Yanaga F, Morimoto S, Harada K, Minakami R, Shiraishi F, Ohta M, Lu QW, Sasaguri T, and Ohtsuki I. Functional consequences of the mutations in human cardiac troponin I gene found in familial hypertrophic cardiomyopathy. J Mol Cell Cardiol 33: 20952107, 2001.[ISI][Medline]
- Towbin JA and Bowles NE. The failing heart. Nature 415: 227233, 2002.[ISI][Medline]
- Tyska MJ, Hayes E, Giewat M, Seidman CE, Seidman JG, and Warshaw DM. Single-molecule mechanics of R403Q cardiac myosin isolated from the mouse model of familial hypertrophic cardiomyopathy. Circ Res 86: 737744, 2000.[Abstract/Free Full Text]
- Van Eyk JE and Hodges RS. The biological importance of each amino acid residue of the troponin I inhibitory sequence 104115 in the interaction with troponin C and tropomyosin-actin. J Biol Chem 263: 17261732, 1988.[Abstract/Free Full Text]
- Van Eyk JE, Thomas LT, Tripet B, Wiesner RJ, Pearlstone JR, Farah CS, Reinach FC, and Hodges RS. Distinct regions of troponin I regulate Ca2+-dependent activation and Ca2+ sensitivity of the acto-S1-TM ATPase activity of the thin filament. J Biol Chem 272: 1052910537, 1997.[Abstract/Free Full Text]