Articles in PresS. Physiol Genomics (May 20, 2003). 10.1152/physiolgenomics.00101.2002
Familial hypertrophic cardiomyopathy mutations in troponin I
(K183∆, G203S, K206Q) enhance filament sliding
Jan Köhler1, Ying Chen2,6, Bernhard Brenner1, Albert M. Gordon3, Theresia Kraft1, Donald A.
Martyn4, Michael Regnier4, Anthony J. Rivera4, Chien-Kao Wang3,4, P. Bryant Chase5
1
Molekular- und Zellphysiologie, Medizinische Hochschule, D-30625 Hannover, Germany
Department of Radiology, University of Washington, Seattle, WA
3
Department of Physiology and Biophysics, University of Washington, Seattle, WA
4
Department of Bioengineering, University of Washington, Seattle, WA
5
Department of Biological Science, Florida State University, Tallahassee, FL
2
6
Present address for Dr. Y. Chen:
UMDNJ RWJ Medical School, Dept. Pathology, Piscataway, NJ 08854
Abbreviated title: C-terminal mutations of cardiac TnI enhance motility
Corresponding author:
P. Bryant Chase, Ph.D., F.A.H.A.
Associate Professor
Florida State University
Department of Biological Science and Program in Molecular Biophysics
Biology Unit One
Tallahassee, FL 32306-4370
850-644-0056
850-644-0481 (fax)
[email protected]
Copyright (c) 2003 by the American Physiological Society.
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ABSTRACT
A major cause of familial hypertrophic cardiomyopathy (FHC) is dominant mutations in cardiac
sarcomeric genes. Linkage studies identified FHC-related mutations in cardiac troponin I’s
(cTnI) C-terminus, 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 (α-MHC or β-MHC) 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 WT cTn. In both trabeculae and motility assays, there was
no loss of inhibition at pCa 9. These results are consistent with TnI’s C-terminus 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.
Keywords: heart, calcium regulation, systole, in vitro motility, troponin exchange
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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 6 mutations in 5 residues, all in the
C-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 C-terminus (43, 44, 47).
Troponin I (I = inhibitory) is crucial for turning myofilaments “off” when [Ca2+]i 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). While 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 C-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
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al. (9) who showed that G203S enhanced Ca2+-sensitivity of force generation but not ATPase
activity, while 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 WT cTnI. In one other
study, Elliott et al. (19) showed that the R162W mutation increased both Ca2+-sensitivity and
also of actin-Tn-Tm-S1 ATPase activity in solution 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 C-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 cTn’s
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
cTn’s 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).
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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, N-terminal Ca2+ binding
site II, cTnC (D65A) (also referred to as xcTnC), by site-directed mutagenesis using the T7-GEN In
Vitro Mutagenesis Kit (USB, Cleveland, OH, USA). A vector pET-24 (Novagen, Madison, WI,
USA) containing the T7 promoter, lac operator, and a kanamycin resistance gene was used for the
expression of the respective WT or mutant clones in E. 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 C-terminal residue numbering is different
between rat and human cTnI because of the additional residue A24 in the N-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).
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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 Cys 39 under denaturing conditions. Labeling was
0.2 moles of rhodamine per mole of xcTnC or 0.4 moles of rhodamine per mole of cTnT in the
respective ternary troponin complexes.
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 –20oC for up to 6 wks; sHMM was stored at 0 – 4oC 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-1 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 0 – 4oC. 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 excess F-actin was added to HMM, followed by
1 mM ATP and ultracentrifugation at 513,000 x g and 4oC (model TLX 120.2, Beckman,
Fullerton, CA). Competent HMM (supernatant) was then diluted to 250 µg ml-1 (determined
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using the Bradford assay due to the presence of ATP and ADP), the concentration used in
motility experiments. Actin and tropomyosin 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 de-ionized 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. 1½ 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-1 BSA in AB to block nonspecific protein binding. After the chamber was flushed
with AB, unlabeled F-actin (~100 µg ml-1, sheared by at least 15 rapid passages through a
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23-guage 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 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 50 – 250 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 F-actin filaments with AB plus 50 – 250 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 MgATP, 1 mM Mg2+, 65 mM Na+ + K+, 10 mM EGTA,
8 - 29 mM propionate, 28 – 70 mM 3-[N-morpholino]propanesulfonic acid (MOPS),
0.085 M ionic strength (Γ/2), 0.5 – 0.75% (w/v) methylcellulose (MC), pH 7.0 at 30oC, the
experimental temperature (20). Ca(propionate)2 was altered to change the pCa (= -log[Ca2+])
between 9.2 – 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 50 – 250 nM each of Tm and Tn. For all motility buffers, 3 mg ml-1
glucose, 100 µg ml-1 glucose oxidase (Sigma, St. Louis, MO), 18 µg ml-1 catalase (Boehringer-
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Mannheim, Indianapolis, IN), and 40 mM DTT (BioRad, Hercules, CA) were added to minimize
photo-oxidation and photobleaching (34). Motility assays at 30oC 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 S.D. 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-1, then the frame-to-frame
speeds 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 of S.D. 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
Inc., Richmond, CA):
su =
s
1 + 10
max
n ( pCa − pCa50 )
+ s min
Eq. 1
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where smax is the speed obtained 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 was of rat hearts obtained as described
previously (22, 57). Adult male Sprague-Dawley rats were euthanized with pentobarbital
(50 mg kg-1) IP. Hearts were rapidly excised, 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 4oC in skinning buffer (in mM: 100 KCl,
10 MOPS, 5 K2EGTA, 9 MgCl2, 4 ATP, pH 7.0 at 4oC, 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, the ends wrapped in
photo-chemically etched T-clips and stored at -20oC 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, Watertown, MA) 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, Japan).
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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 units ml-1 creatine kinase (CK, Sigma, St. Louis, MO), 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 Ca(propionate)2 (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 4 wells could be
independently maintained by Peltier thermo-electric chips controlled by an ATR-4 adaptable
thermo-regulator (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 + S.D., 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 re-stretched 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 1 by nonlinear least squares regression (SigmaPlot, SPSS Inc., Richmond, CA) to
the Hill equation (Eq. 2):
PG-00101-2002 – Köhler et al. – C-terminal mutations of cardiac TnI enhance motility (5/19/2003)
F=
Fmax
1 + 10 n ( pCa − pCa50 )
Page 12
Eq. 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 (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-1 cTn) at 10oC 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 15oC 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
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cardiac and skeletal muscle preparations following TnC replacement with 5’ IATR labeled TnC
(41, 42). To avoid non-specific binding of fluorescently-labeled cTn, the exchange protocol was
modified by incubating trabeculae with 1 mg ml-1 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 for 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 BioRad MRC-600
laser-scanning confocal microscope.
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RESULTS
Unloaded sliding of single actin filaments
To test the effects of mutant cTnI’s on actomyosin function, we first investigated sliding of
isolated, well-regulated RhPh-actin filaments in an in vitro motility assay. Fig. 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
224 – 2419 uniformly moving filament paths that could be unambiguously tracked for at least 2 s
(Methods). Assays conducted with either WT cTn (open circles) or with cTn reconstituted from
WT cTnC, WT cTnT and cTnI,K206Q (solid squares) 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).
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 (open bars in Fig. 2A). The
concentration of mutant cTn’s 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 47 – 61% above that for WT (hatched bars in Fig. 2A), and also
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above the average sliding speed for unregulated F-actin (“unreg”) 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).
It is possible that cTnI mutations could affect regulated motility assays differently when HMM
from α- or β-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 α-MHC) or from hearts from PTU-treated rats (predominantly β-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-1 (N = 8) with rabbit skeletal HMM, 1.40 + 0.51 µm s-1
(N = 5) with untreated rat cardiac HMM, and 1.02 + 0.03 µm s-1 (N = 2) with PTU-treated rat
cardiac HMM. The high [Ca2+] data with rabbit skeletal HMM from Fig. 2A (hatched bars) was
replotted in Fig. 3A after normalization to WT. Fig. 3B, C illustrates that a similar pattern was
obtained with both α- and β-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 – 2.4-fold.
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 permeablized
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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 photo-bleaching 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.
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-1. At high magnification, fluorescent bands at the periphery of
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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 minutes
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. 4E, F. This pattern suggests that cTn binding is specific to thin
filaments and that non-specific binding is minimal. We therefore chose 2 hr 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 N-terminal Met residues combined
with the absence of an acetylated N-terminus, absence of phosphorylated residues, and presence
of only a single, adult isoform of cTnT. Fig. 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,
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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 5 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.
Recombinant cTn(Rh-cTnC,D65A). After demonstrating that exogenous cTn can be incorporated
into permeablized 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 & II) of sTnC, xxsTnC (58). Rat xcTnC is comparable to mutant CBMII 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. 6B – D). Co-localization of Rh-labeled cTn (Fig. 7C) and phalloidin-greenlabeled actin (Fig. 6B) is clearly evident in the average sarcomere scans of Figs. 6B and C
(Fig. 6D).
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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, 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.
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DISCUSSION
In this study, we found that: 1. In comparison 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 α-MHC), or cardiac muscle
from PTU-treated rats (primarily β-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
C-terminus of TnI in determining the duration of acto-myosin 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
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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 4, 5, 6 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 hrs, 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 the exchange buffer (Methods).
Changes in myofilament function due to cTnI mutations. The three C-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, 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
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in solution) is consonant with the studies of C-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, 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. 1 – 3) were not also observed in force-pCa relations of trabeculae
(Fig. 7). There is a related example of such disparate effects on isometric force versus 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 7 - 50% (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
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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 versus 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 cTn’s 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 cTn’s is
reminiscent of the effect of sTn (20) and supports the suggestion that regulatory proteins can
uniquely modulate the kinetics of interaction of myosin crossbridges 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
versus 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
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is slow (g1x/h in the framework of Ref. 27, where x is the displacement between a cross-bridge’s
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 crossbridge 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 C-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
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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 C-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, α−Tm,D175N, α−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. 1 – 3 and Refs. 2, 26, 37, 66).
Furthermore, the majority of FHC-related mutations in cTnT (32), cTnI (Figs. 1, 2 and Refs. 9,
19, 35, 67) and α-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).
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Interestingly, recent examinations of myosin heavy chain (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 β-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 if there is a common underlying route to
hypertrophy for the mutations identified in a wide variety of sarcomeric proteins.
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ACKNOWLEDGEMENTS
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; Dr. Larry S. Tobacman and Earl Homsher for
generous assistance in preliminary experiments. Funding: NIH HL63974, HL52558, NS08384,
American Heart Association – Washington State Affiliate, University of Washington Royalty
Research Fund, Biomedical Science Exchange Program and the Hannover Medical School.
M. Regnier is and Established Investigator of the American Heart Association.
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FIGURE LEGENDS
Figure 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 HMM, actin and Tm with WT 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 = 224 – 2419 filament paths per flow cell which correspond 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 shows video image of regulated, RhPh-labeled actin filaments at pCa 5.
Figure 2. In vitro motility speed-pCa regression parameter estimates in the presence of 50 –
250 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 panel A (“unreg”).
Nonlinear least squares regression was used to fit in vitro motility data were 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 conducted with rabbit skeletal HMM.
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Figure 3. Maximum speed of in vitro motility with (A) rabbit skeletal HMM, (B) rat cardiac
HMM (predominantly α-MHC isoform) and (B) rat cardiac HMM prepared from PTU-treated
animals (predominantly β-MHC isoform). Assays with regulated actin contained Tm and either
WT cTnI, cTnI,K183∆, cTnI,G203S, or cTnIK206Q. 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-1 (N = 8) with rabbit skeletal HMM, 1.40
+ 0.51 µm s-1 (N = 5) with rat cardiac HMM, and 1.02 + 0.03 µm s-1 (N = 2) with PTU-rat
cardiac HMM.
Figure 4. Incorporation of exogenous troponin into permeablized cardiac trabeculae. For the
experiments shown in this figure, 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 hrs.
Points are single determinations and line is nonlinear least squares regression fit to y = 1 – e-t/τ.
(B – F) Confocal microscope images of cardiac trabeculae incubated for 15 min (B, C), 90 min
(D – E) or 120 min (F) with 5’ IATR-labeled cTn. Scale bars represent 10 µm (B, D) and 2 µm
(C, E, F). Insets in panels B and D show florescence intensity profiles across diameter of the
trabecular preparations.
Figure 5. Incorporation of recombinant WT cTn into permeablized rat cardiac trabeculae.
(A) Slow time-base recording of force. Activation pCa indicated below record; relaxing pCa 9
otherwise. Transients in the force record occur at 5 s intervals due to periodic
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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 steadystate, 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 mean ± SEM (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 shows small reduction in maximum isometric force
to 86% of initial control at pCa 4.
Figure 6. Functional validation of exogenous troponin exchange into cardiac trabeculae. For
the experiments shown in this figure, 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 that 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, C) Confocal microscope images showing co-localization of (B) Ph-green (actin) and (C) RhxcTnC. (D) Fluorescence intensity scans of Ph-green (green) and Rh-xcTnC (red) images.
Fluorescence intensity was averaged over the region delimited by white, dotted lines in panel C
and the equivalent region in panel B. Fluorescence intensity is in arbitrary units and the Ph-
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green scan was offset relative to the Rh-xcTnC scan for clarity. Length axis for panel D applies
to panels B – D.
Figure 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 mean ± SEM (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 shows reduction in
maximum isometric force (pCa 4) after exchange to 86% of the pre-exchange value for WT and
to 84% for cTnI,K206Q.
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BIBLIOGRAPHY
1. Backx PH, Gao W-D, 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: 487-500, 1994.
2. Bing W, Redwood CS, Purcell IF, Esposito G, Watkins H, and Marston SB. Effects of
two hypertrophic cardiomyopathy mutations in α-tropomyosin, Asp175Asn and Glu180Gly, on
Ca2+ regulation of thin filament motility. Biochem Biophys Res Commun 236: 760-764, 1997.
3. Bing W, Knott A, Redwood C, Esposito G, Purcell I, Watkins H, and Marston S. Effect
of hypertrophic cardiomyopathy mutations in human cardiac muscle α-tropomyosin (Asp175Asn
and Glu180Gly) on the regulatory properties of human cardiac troponin determined by in vitro
motility assay. J Mol Cell Cardiol 32: 1489-1498, 2000.
4. Bonne G, Carrier L, Richard P, Hainque B, and Schwartz K. Familial hypertrophic
cardiomyopathy: from mutations to functional defects. Circ Res 83: 580-593, 1998.
5. Brandt PW, Diamond MS, Rutchik JS, and Schachat FH. Co-operative interations
between troponin-tropomyosin units extend the length of the thin filament in skeletal muscle. J
Mol Biol 195: 885-896, 1987.
6. Brenner B. Technique for stabilizing the striation pattern in maximally calcium-activated
skinned rabbit psoas fibers. Biophys J 41: 99-102, 1983.
7. Brenner B, and Eisenberg E. Rate of force generation in muscle: correlation with
actomyosin ATPase activity in solution. Proc Natl Acad Sci USA 83: 3542-3546, 1986.
8. 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-
PG-00101-2002 – Köhler et al. – C-terminal mutations of cardiac TnI enhance motility (5/19/2003)
Page 33
labeled troponin I incorporated into skinned fibers of rabbit psoas muscle. Biophys J 77: 26772691, 1999.
9. 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: 443-451, 2002.
10. Chase PB, and Kushmerick MJ. Effects of pH on contraction of rabbit fast and slow
skeletal muscle fibers. Biophys J 53: 935-946, 1988.
11. 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: 231-246, 1993.
12. 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: 119-129,
1994.
13. 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.
14. Chase PB, Köhler J, Chen Y, Wang C-K, Luo Z, Kraft T, and Brenner B. Physiological
effects of cardiac troponin I mutations related to familial hypertrophic cardiomyopathy. Biophys
J 80: 342a-343a, 2001.
15. Chase PB. Proposed mechanism of hypertrophy due to C-terminal mutations in cardiac
troponin I. The Physiologist 45: 71, 2002.
PG-00101-2002 – Köhler et al. – C-terminal mutations of cardiac TnI enhance motility (5/19/2003)
Page 34
16. Chomczynski P, and Sacchi N. Single-step method of RNA isolation by acid guanidinium
thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156-159, 1987.
17. Dong W, Rosenfeld SS, Wang C-K, 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.
18. Ebus JP, and Stienen GJM. Effects of 2,3-butanedione monoxime on cross-bridge kinetics
in rat cardiac muscle. Pflügers Arch 432: 921-929, 1996.
19. Elliott K, Watkins H, and Redwood CS. Altered regulatory properties of human cardiac
troponin I mutants that cause hypertrophic cardiomyopathy. J Biol Chem 275: 22069-22074,
2000.
20. Gordon AM, LaMadrid M, Chen Y, Luo Z, and Chase PB. Calcium regulation of skeletal
muscle thin filament motility in vitro. Biophys J 72: 1295-1307, 1997.
21. Gordon AM, Homsher E, and Regnier M. Regulation of Contraction in Striated Muscle.
Physiol Rev 80: 853-924, 2000.
22. Hancock WO, Martyn DA, Huntsman LL, and Gordon AM. Influence of Ca2+ on force
redevelopment kinetics in skinned rat myocardium. Biophys J 70: 2819-2829, 1996.
23. 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: 1022-1027, 1991.
24. 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: 985-993, 1992.
PG-00101-2002 – Köhler et al. – C-terminal mutations of cardiac TnI enhance motility (5/19/2003)
Page 35
25. Homsher E, Kim B, Bobkova A, and Tobacman LS. Calcium regulation of thin filament
movement in an in vitro motility assay. Biophys J 70: 1881-1892, 1996.
26. 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: 233-243, 2000.
27. Huxley AF. Muscle structure and theories of contraction. Prog Biophys 7: 255-318, 1957.
28. 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: 1447-1455,
1996.
29. 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: 805-811, 2000.
30. 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
α-tropomyosin mutation (V95A) is associated with mild cardiac phenotype, abnormal calcium
binding to troponin, abnormal myosin cycling, and poor prognosis. Circulation 103: 65-71,
2001.
31. Kimura A, Harada H, Park J-E, Nishi H, Satoh M, Takahashi M, Hiroi S, Sasaoka T,
Ohbuchi N, Nakamura T, Koyanagi T, Hwang T-H, Choo J-A, Chung K-S, 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: 379-382, 1997.
PG-00101-2002 – Köhler et al. – C-terminal mutations of cardiac TnI enhance motility (5/19/2003)
Page 36
32. Knollmann BC, and Potter JD. Altered regulation of cardiac muscle contraction by
troponin T mutations that cause familial hypertrophic cardiomyopathy. Trends Cardiovasc Med
11: 206-212, 2001.
33. 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: 3557-3562, 2002.
34. Kron SJ, Toyoshima YY, Uyeda TQP, and Spudich JA. Assays for actin sliding
movement over myosin-coated surfaces. Methods Enzymol 196: 399-416, 1991.
35. 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: 11670-11678, 2002.
36. 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:
Submitted, 2002.
37. 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: 2842-2848, 1996.
38. Malik MSA, and Watkins H. The molecular genetics of hypertrophic cardiomyopathy. Curr
Opin Cardiol 12: 295-302, 1997.
39. Maron BJ. Hypertrophic cardiomyopathy. Lancet 350: 127-133, 1997.
40. 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: 1984-1993, 1994.
PG-00101-2002 – Köhler et al. – C-terminal mutations of cardiac TnI enhance motility (5/19/2003)
Page 37
41. 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:
1480-1493, 1999.
42. 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.
43. 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: I618, 1999.
44. 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: 521-525, 2000.
45. 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: 20245-20251,
2001.
46. 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: 585-600,
1985.
47. 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: 446-451, 2002.
PG-00101-2002 – Köhler et al. – C-terminal mutations of cardiac TnI enhance motility (5/19/2003)
Page 38
48. 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: 63-74, 1997.
49. 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: 609-620, 2000.
50. Pardee JD, and Spudich JA. Purification of muscle actin. Methods Enzymol 85: 164-181,
1982.
51. Perry SV. Troponin T: genetics, properties and function. J Muscle Res Cell Motil 19: 575602, 1998.
52. Potter JD. Preparation of troponin and its subunits. Methods Enzymol 85: 241-263, 1982.
53. Putkey JA, Sweeney HL, and Campbell ST. Site-directed mutation of the trigger calciumbinding sites in cardiac troponin C. J Biol Chem 264: 12370-12378, 1989.
54. Rarick HM, Tu X-H, 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:
26887-26892, 1997.
55. Redwood CS, Moolman-Smook JC, and Watkins H. Properties of mutant contractile
proteins that cause hypertrophic cardiomyopathy. Cardiovasc Res 44: 20-36, 1999.
56. Regnier M, Martyn DA, and Chase PB. Calmidazolium alters Ca2+ regulation of tension
redevelopment rate in skinned skeletal muscle. Biophys J 71: 2786-2794, 1996.
57. Regnier M, Rivera AJ, Chen Y, and Chase PB. 2-deoxy ATP enhances contractility of rat
cardiac muscle. Circ Res 86: 1211-1217, 2000.
PG-00101-2002 – Köhler et al. – C-terminal mutations of cardiac TnI enhance motility (5/19/2003)
Page 39
58. Regnier M, Rivera AJ, Bates MA, Wang C-K, Chase PB, and Gordon AM. Thin
filament near-neighbor regulatory unit interactions affect rabbit skeletal muscle steady state
force-Ca2+ relations. J Physiol 540: 485-497, 2002.
59. Sellers JR, Cuda G, Wang F, and Homsher E. Myosin-specific adaptations of the motility
assay. Methods Cell Biol 39: 23-49, 1993.
60. 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: 737-745, 2000.
61. Shiraishi F, Kambara M, and Ohtsuki I. Replacement of troponin components in
myofibrils. J Biochem 111: 61-65, 1992.
62. Smillie LB. Preparation and identification of α- and β-tropomyosins. Methods Enzymol 85:
234-241, 1982.
63. Solaro RJ, and Rarick HM. Troponin and tropomyosin: proteins that switch on and tune in
the activity of cardiac myofilaments. Circ Res 83: 471-480, 1998.
64. 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: 229-234, 1992.
65. Sweeney HL, Corteselli SA, and Kushmerick MJ. Measurements on permeabilized
skeletal muscle fibers during continuous activation. Am J Physiol 252: C575-C580, 1987.
66. 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: 14406-14410, 1998.
67. 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
PG-00101-2002 – Köhler et al. – C-terminal mutations of cardiac TnI enhance motility (5/19/2003)
Page 40
troponin I gene found in familial hypertrophic cardiomyopathy. J Mol Cell Cardiol 33: 20952107, 2001.
68. Towbin JA, and Bowles NE. The failing heart. Nature 415: 227-233, 2002.
69. Tyska MJ, Hayes E, Giewat M, Seidman CE, Seidman JG, and Warshaw DM. Singlemolecule mechanics of R403Q cardiac myosin isolated from the mouse model of familial
hypertrophic cardiomyopathy. Circ Res 86: 737-744, 2000.
70. Van Eyk JE, and Hodges RS. The biological importance of each amino acid residue of the
troponin I inhibitory sequence 104-115 in the interaction with troponin C and tropomyosin-actin.
J Biol Chem 263: 1726-1732, 1988.
71. 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.
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In vitro motility
-1
filament sliding speed (µm s )
Figure 1
8
6
4
2
0
9
8
7
pCa
6
5
PG-00101-2002 – Köhler et al. – C-terminal mutations of cardiac TnI enhance motility (5/19/2003)
In vitro motility
-1
filament sliding speed (µm s )
Figure 2
A
6
pCa > 8
pCa < 5
**
**
**
G203S
K206Q
4
2
0
unreg
WT
6.6
In vitro motility
pCa50
6.4
K183∆
**
B
**
6.2
**
6.0
5.8
5.6
0.0
WT
K183∆
cTnI
G203S
K206Q
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Figure 3
Rat cardiac HMM
Rabbit skeletal HMM
speed (normalized)
2.5
A
PTU-treated rat cardiac HMM
B
C
2.0
**
1.5
**
**
**
**
**
*
1.0
*
0.5
0.0
WT
K183∆ G203S K206Q
WT
K183∆ G203S K206Q
cTnI
WT
K183∆ G203S K206Q
PG-00101-2002 – Köhler et al. – C-terminal mutations of cardiac TnI enhance motility (5/19/2003)
A
Fluorescence intensity (normalized)
Figure 4
1.00
0.75
0.50
0.25
0.00
0
30
60
90
Time (min)
B
C
D
E
F
120
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Figure 5
A
C
1
0.50
pre
0.25
post
0.75
Fmax
1.00
Force (normalized)
B
0
0.00
6.0
5.5
5.0
pCa
4.5
4.0
PG-00101-2002 – Köhler et al. – C-terminal mutations of cardiac TnI enhance motility (5/19/2003)
Figure 6
cTn (Rh-cTnC, D65A)
exchange
a
pCa
9.0
4.0
9.0
9.0
4.0
9.0
b
c
d
0
2
4
6
8
10
12
14
x (um )
16
18
20
22
24
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Figure 7
1
0.75
max
0.50
0
0.00
6.0
5.5
5.0
pCa
WT
4.5
post
pre
pre
0.25
post
F
Force (normalized)
1.00
K206Q
4.0