2104 Biophysical Journal Volume 105 November 2013 2104–2113

Instability in the Central Region of Tropomyosin Modulates the Function of

Its Overlapping Ends

Ranganath Mamidi,† Mariappan Muthuchamy,‡ and Murali Chandra†*

†
Department of Integrative Physiology and Neuroscience, College of Veterinary Medicine, Washington State University, Pullman, Washington;
and ‡Department of Medical Physiology, Texas A&M Health Science Center, College of Medicine, College Station, Texas

ABSTRACT The causal link between disparate tropomyosin (Tm) functions and the structural instability in Tm is unknown. To
test the hypothesis that the structural instability in the central region of Tm modulates the function of the overlapping ends of
contiguous Tm dimers, we used transgenic mice (TmDM) that expressed a mutant a-Tm in the heart; S229E and H276N sub-
stitutions induce structural instability in the central region and the overlapping ends of Tm, respectively. In addition, two mouse
cardiac troponin T mutants (TnT1–44D and TnT45–74D) that have a divergent effect on the overlapping ends of Tm were employed.
The S229E-induced instability in the central region of TmDM altered the overlapping ends of TmDM, thereby it negated the atten-
uating effect of H276N on Ca2þ-activated maximal tension. The rate of cross-bridge detachment (g) decreased in TmDMþTnTWT
and TmH276NþTnTWT fibers but increased in TmDMþTnT45–74D fibers; however, TnT45–74D did not alter g, demonstrating that
S229E in TmDM had divergent effects on g. The S229E substitution in TmDM ablated the H276N-induced desensitization of
myofilament Ca2þ sensitivity in TmDMþTnT1–44D fibers. To our knowledge, novel findings from this study show that the structural
instability in the central region of Tm modifies cardiac contractile function via its effect on the overlapping ends of contiguous Tm.

INTRODUCTION
A structural feature that is common to all types of tropomy-
osin (Tm) includes the seven quasi-repeating regions of 40
aa (1–6). However, naturally occurring amino acid substitu-
tions in Tm isoforms lead to nonuniformity in different re-
gions of Tm (2,4,7–10). A consequence of amino acid
substitutions in various Tm isoforms is that such substitu-
tions have the potential to induce local structural instability,
an effect that introduces conformational plasticity or flexi-
bility in different regions of Tm. Conformational flexibility
in Tm is now considered essential for promiscuity that
yields disparate Tm functions (10–14). However, the rela-
tionship between structural instability in Tm and varied car-
diac contractile function remains poorly understood.
The flexibility of Tm is also significantly modified by
troponin T (TnT). Not only does TnT affect the flexibility
of Tm, but it also modulates how Tm interacts with actin
(15–17). Using a transgenic (TG) mouse model, we showed
that the interplay between the N-terminus of cardiac TnT
(TnT) and the overlapping ends of Tm resulted in different
states of Tm on the actin filament (16). The H276N substi-
tution in a-Tm (TmH276N)—which destabilizes the overlap-
ping ends of Tm (18)—did not affect Ca2þ-activated
maximal tension but slowed the cross-bridge (XB) detach-
ment rate (g) (16). Furthermore, we previously demon-
strated that TmH276N attenuated Ca2þ-activated maximal
tension in muscle fibers containing a mouse cardiac TnT
deletion mutant in which 1–44 aa were deleted (TnT1–44D)
and in muscle fibers containing a mouse cardiac TnT dele-
tion mutant where 45–74 aa were deleted (TnT45–74D) (16).
Submitted June 4, 2013, and accepted for publication September 17, 2013.
*Correspondence: murali@vetmed.wsu.edu
Editor: KW Ranatunga.
Ó 2013 by the Biophysical Society
0006-3495/13/11/2104/10 $2.00
Ca2þ-mediated activation of the thin filament is also likely
to be modified by the central region of Tm (145–235) because
it interacts with the Tn complex (5,19,20). Thus, any struc-
tural instability or subtle conformational changes in the cen-
tral region of the filamentous Tm may be transduced to the
terminal ends of Tm to modulate contractile function. For
example, the substitution of a negatively charged amino
acid at position 229 (S229E) of Tm not only increased the dis-
tance between two helically intertwined strands of Tm, but
also affected cardiac contractility as evidenced by decreased
rates of pressure development and relaxation in work-per-
forming hearts (18). Interestingly, b-Tm from striated muscle
contains the naturally occurring S229E substitution (18,21).
Results from previous TG mouse studies showed that expres-
sion of b-Tm in the cardiac muscle prolonged the time to half
relaxation, reduced the rate of relaxation in work-performing
hearts (22), increased myofilament Ca2þ sensitivity, and
attenuated the rate of relaxation in isolated cardiac myocytes
(23). Collectively, these observations suggest that the charge-
induced instability in the central region of Tm may be one of
the key determinants of Tm isoform-mediated differences in
contractile function. However, the molecular mechanism by
which the structural instability in the central region of Tm
modulates contractile function remains unknown.
The purpose of this study was to understand how the struc-
tural instability in the central region of Tm modulated cardiac
contractile function. We used cardiac muscle fibers from
a TG mouse model (TmDM) that expressed 88% of a mutant
form of a-Tm that contained S229E and H276N substitutions
in the mouse cardiac a-Tm. We also used TnT1–44D and
TnT45–74D proteins that interact with the overlapping ends
of contiguous Tm. Previous research demonstrated that
TnT1–44D and TnT45–74D altered the H276N-mediated effects
http://dx.doi.org/10.1016/j.bpj.2013.09.026
S229E Affects the Overlapping Ends of Tropomyosin
on the overlapping ends of TmH276N (16). The TmDM TG
mouse model enabled us to test if the S229E substitution
in TmDM modulated the H276N, TnT1–44D, or TnT45–74D-
mediated effects on the overlapping ends of TmDM (schemat-
ically represented in Fig. 1). A novel finding (to our
knowledge) in this study showed that the S229E substitution
in TmDM altered the TnT45–74D-mediated effect on Ca2þ-
activated maximal tension. The S229E substitution in
TmDM decreased g in TnTWT reconstituted fibers but
increased g in TnT45–74D reconstituted fibers. Another signif-
icant finding was that the S229E substitution in TmDM abla-
ted the attenuating effect of TnT1–44D on myofilament Ca2þ
sensitivity. Results from this study showed that the S229E
substitution in the central region of TmDM modulated the
overlapping-end-mediated effects of Tm on various contrac-
tile parameters. To our knowledge, novel findings in this
study provide new evidence for a mechanistic understanding
of the causal link between innate structural instability within
Tm and disparate Tm functions.
MATERIALS AND METHODS
Animal handling
TG mice (FVB/N strain), which expressed ~88% of TmDM in the myocar-
dium, were previously generated and characterized (18). Nontransgenic
mice (FVB/N strain), which contained the endogenous wild-type (WT)
a-Tm (TmWT) in the myocardium, were used as controls. All animals
used in this study received proper care and treatment. All experiments
were carried out in accordance with the guidelines laid down by the Wash-
ington State University Institutional Animal Care and Use Committee.
Preparation of detergent-skinned cardiac muscle
fibers
Details regarding the preparation of detergent-skinned papillary muscle
fiber bundles from the hearts of nontransgenic and TG mice are mentioned
in the Supporting Material.
Expression and purification of recombinant
mouse cardiac Tn subunits
Recombinant c-myc-tagged mouse cardiac TnT (c-myc TnT); TnT1–44D, in
which the first 44 aa were deleted; TnT45–74D, in which 45–74 aa were
deleted; mouse cardiac TnI (TnI); and mouse cardiac TnC (TnC) were
expressed in BL21*DE3 cells (Novagen, Madison, WI) and purified as
TnC TnT 1-44
CR of TnT
TnT 45-74
TnI
C N
Tm-Tm
overlap region
H276N S229E Actin
complex is shown as described previously (48). Because the local instability in Tm does not affect the binding of Tm to Tn (41), and residues 1–77 of
cTnT are not known to interact with Tm (47), it is likely that the S229E substitution in Tm affects myofilament function via a long-range conformational
effect on the overlapping ends of Tm.
2105
described earlier (17,24). The purity of protein fractions was evaluated
using a 12.5% SDS PAGE. Pure protein fractions were pooled and thor-
oughly dialyzed against deionized water containing 15 mM b-mercaptoe-
thanol, lyophilized, and stored at 80 C for long-term use.
Reconstitution of recombinant Tn subunits into
detergent-skinned cardiac muscle fibers
The protocol for reconstitution of Tn subunits into detergent-skinned cardiac
fibers is described in the Supporting Material. For clarity and simplicity,
the following terminologies are used for the reconstituted fibers: TmWT
fibers reconstituted with TnTWT þ TnI þ TnC are referred to as TmWT
þTnTWT fibers; TmWT fibers reconstituted with TnT1–44DþTnIþTnC are
referred to as TmWTþTnT1–44D fibers; TmWT fibers reconstituted with
TnT45–74DþTnIþTnC are referred to as TmWTþTnT45–74D fibers; TmDM
fibers reconstituted with TnTWTþTnIþTnC are referred to as TmDM
þTnTWT fibers; TmDM fibers reconstituted with TnT1–44DþTnIþTnC are
referred to as TmDMþTnT1–44D fibers; and TmDM fibers reconstituted with
TnT45–74DþTnIþTnC are referred to as TmDMþTnT45–74D fibers.
SDS-PAGE and Western blot analysis of
reconstituted muscle fibers
Details regarding gel preparations, solubilization of muscle fibers, and
Western blotting are mentioned in the Supporting Material.
Simultaneous measurement of steady-state
isometric force and ATPase activity
The muscle fiber was attached between a motor arm (322C, Aurora Scien-
tific, Aurora, Ontario, Canada) and a force transducer (AE 801, Sensor One
Technologies, Sausalito, CA) using T-shaped aluminum clips. The sarco-
mere length (SL) of the muscle fiber was set to 2.3 mm under relaxing con-
ditions (pCa 9.0; pCa ¼ log of [Ca2þ]free) using a laser light diffraction
pattern (25,26). The muscle fiber was subjected to two cycles of maximal
activation and relaxation at 20 C and the SL was readjusted to 2.3 mm if
needed. The muscle fiber was immersed in a constantly stirred chamber
containing Ca2þ solutions of concentrations ranging from pCa 4.3 to 9.0.
The pH of the Ca2þ solutions was adjusted to 7.0 using KOH. The ionic
strength was 180 mM. Simultaneous measurement of steady-state isometric
force and ATPase activity was according to the procedures described earlier
(26–29). Other details are provided in the Supporting Material.
Measurement of the rate of force redevelopment
(ktr)
Measurement of ktr in maximally activated (pCa 4.3) muscle fiber was ac-
cording to the modified protocol described by Brenner and Eisenberg (30).
FIGURE 1 Schematic representation of S229E
and H276N substitutions in TmDM. S229E and
H276N substitutions in Tm are represented by
Tm
black stars and black filled circles, respectively.
The central helical region of cTnT (residues 78–
193) is denoted by CR. The inter-molecular inter-
action between the head-to-tail overlapping region
of contiguous Tm dimers and the N-terminus of
cTnT is denoted by the double-headed arrow
(adapted from 47) The orientation of the Tn
Biophysical Journal 105(9) 2104–2113
2106
During the steady-state activation, the muscle fiber was quickly (within
1 ms) slackened by 10% of the muscle length, using a high-speed length-
control device (Aurora Scientific). After a shortening period of 25 ms,
the muscle fiber was quickly stretched (for 1 ms) beyond its original length
by 10% to ensure that any remaining XBs were mechanically detached and
that the residual force was not > ~10% of the initial steady-state isometric
force. ktr was determined by fitting the following monoexponential equation
to the force redevelopment.

B
F ¼ ðFss  Fres Þ 1  ektr t þ Fres ;
where F is the force at time t, Fss is the steady-state isometric force, and Fres
is the residual force from which the force redevelops.
Data analysis
Data were analyzed using a two-way analysis of variance (ANOVA). One
factor was Tm variant (TmWT or TmDM) and the other factor was TnT
variant (TnTWT or TnT1–44D or TnT45–74D). Thus, we employed a two-
way ANOVA to test the hypothesis that the functional impact of Tm var-
iants on a given contractile parameter was modulated by the TnT variant
(interaction effect). When the interaction effect was significant, we
observed that the functional impact of TmWT and TmDM on TnTWT,
TnT1–44D, or TnT45–74D were dissimilar. When the interaction effect was
not significant, we interpreted the main effects due to either Tm variants
or TnT variants. To test the functional impact of Tm or TnT variants on
various contractile parameters, post hoc multiple pairwise comparisons
were performed using the uncorrected Fisher’s least significant difference
(LSD) method. To estimate pCa50 (log of [Ca2þ]free required for half-
maximal activation) and the Hill coefficient (nH), normalized pCa-tension
data were fitted to the Hill equation. Data presented here for TmWT fibers
were from our recently published study (16). Values are reported as
mean 5 SE. The criterion for statistical significance was set at P < 0.05.
RESULTS
Western blot analysis of reconstituted muscle
fibers
The c-myc-tagged TnT (TnTWT) served as the control. In the
Tn reconstitution protocol, the exogenously added Tn com-
plex consisted of TnT (TnTWT or TnT1–44D or TnT45–74D),
TnI, and TnC proteins. We have previously shown that the
endogenous Tn complex is replaced as a whole when a
vast excess of exogenously added TnT competes with the
endogenous TnT (15). Thus, the extent of incorporation of
TnTWT or TnT deletion mutants could be used as an index
of the extent of replacement of the endogenous Tn complex.
The differential migration pattern of the c-myc-tagged TnT,
when compared to the endogenous TnT, enabled us to assess
the incorporation of TnTWT. The incorporation of TnT1–44D
or TnT45–74D proteins was assessed based on their differen-
tial gel migration pattern and Western blot analysis. The
extent of incorporation was estimated by dividing the opti-
cal band intensity of the exogenously added TnT with the
total band intensity (for details, see the Supporting Mate-
rial). In the TmWT fiber groups, the level of incorporation
was as follows: 93% for TnTWT (lane 2 of panel A in
Fig. 2); 100% for TnT1–44D (lane 3 of panel A in Fig. 2);
and 87% for TnT45–74D (lane 4 of panel A in Fig. 2). In
Biophysical Journal 105(9) 2104–2113
Mamidi et al.
A
FIGURE 2 Western blot analysis of reconstituted cardiac muscle fibers.
Reconstituted muscle fibers were solubilized in 2% SDS (49). Solubilized
fiber samples were electrophoresed on a 10% SDS gel. Protein bands
were transferred to a PVDF membrane for Western blot analysis. TnTWT,
TnT1–44D, and TnT45–74D were identified using a monoclonal anti-TnT pri-
mary antibody. Panels A and B indicate samples from TmWT and TmDM
fibers, respectively. Lane 1 shows purified recombinant cmyc-tagged
TnTWT and TnT. Lanes 2, 3, and 4 show samples from cmyc-tagged TnTWT,
TnT1–44D, and TnT45–74D reconstituted fibers, respectively.
the TmDM fiber groups, the level of incorporation was as
follows: 82% for TnTWT (lane 2 of panel B in Fig. 2);
100% for TnT1–44D (lane 3 of panel B in Fig. 2); and 76%
for TnT45–74D (lane 4 of panel B in Fig. 2).
Effect of TmDM on Ca2D-activated maximal
tension in TnT1–44D or TnT45–74D reconstituted
cardiac muscle fibers
Charge-mediated (S229E) structural instability in the cen-
tral region of Tm alters cardiac contractile activation at
the myofilament and whole heart levels (18). However, the
molecular basis for the effect of S229E on contractile func-
tion remains unknown. Here, we tested the hypothesis that
the S229E substitution in TmDM altered how TnT1–44D
or TnT45–74D mutants affected the overlapping ends
of TmDM. Previously, we showed that TnT1–44D and
TnT45–74D modulated the effects of TmH276N on contractile
function; the H276N substitution in TmH276N altered the
structure and function of the overlapping ends of contiguous
TmH276N (16). Thus, any changes induced by TmDM in
TnT1–44D or TnT45–74D reconstituted fibers could be directly
ascribed to the impact of the S229E substitution on the
overlapping ends of TmDM. We measured how TmDM
affected Ca2þ-activated maximal tension (pCa 4.3) in
TnT1–44D or TnT45–74D reconstituted cardiac muscle fibers.
Two-way ANOVA revealed a significant interaction effect
(P < 0.05), suggesting that TmDM modified the impact of
TnT1–44D and TnT45–74D on Ca2þ-activated maximal
tension.
To identify the factor responsible for the interaction ef-
fect, we performed subsequent post hoc tests using multiple
S229E Affects the Overlapping Ends of Tropomyosin
pairwise comparisons (uncorrected Fischer’s LSD test). Post
hoc analysis revealed that Ca2þ-activated maximal tension
decreased in TnT1–44D reconstituted fibers, whether the
Tm background was TmWT or TmDM (Fig. 3). Specifically,
Ca2þ-activated maximal tension was attenuated by ~36%
in TmWTþTnT1–44D fibers and 21% in TmDMþTnT1–44D
fibers (Fig. 3). On the other hand, Ca2þ-activated maximal
tension remained unaffected in TmDMþTnT45–74D fibers
(Fig. 3) or TmDMþTnTWT fibers (Table 1). Interestingly,
Ca2þ-activated maximal tension decreased significantly in
TmH276NþTnT45–74D fibers (16). Previously, we demon-
strated that TnT45–74D did not alter Ca2þ-activated maximal
tension in fibers containing TmWT, but attenuated Ca2þ-acti-
vated maximal tension in the presence of TmH276N (16).
Therefore, our current finding that Ca2þ-activated maximal
tension remained unaffected in TmDMþTnT45–74D fibers
(Fig. 3) indicated that the S229E-induced instability in the
central region of TmDM significantly altered the overlapping
ends of TmDM. This observation is further substantiated
by the finding that S229E minimized—although not
completely—the attenuating effect of TnT1–44D on Ca2þ-
activated maximal tension in TmDMþTnT1–44D fibers
(Fig. 3).
Effect of TmDM on Ca2D-activated maximal
ATPase activity in TnT1–44D or TnT45–74D
reconstituted cardiac muscle fibers
To further test whether TmDM affected maximal ATPase
activity, we measured Ca2þ-activated maximal ATPase
activity in reconstituted muscle fibers. In the presence of
TmWT, values of ATPase activity (in pmol$mm3$s1)
were as follows: 304.38 5 9.36, 225.33 5 10.67, and
303.79 5 7.79 for TnTWT, TnT1–44D, and TnT45–74D recon-
FIGURE 3 Effect of TmWT and TmDM on Ca2þ-activated maximal ten-
sion in TnT1–44D or TnT45–74D reconstituted cardiac muscle fibers. Ca2þ-
activated maximal tension (pCa 4.3) was measured at 20 C. The resting
SL was 2.3 mm. Two-way ANOVA revealed a significant Tm-cTnT interac-
tion effect (P < 0.05), indicating that TmDM altered the effects of TnT1–44D
and TnT45–74D on maximal tension. Post hoc multiple pairwise (Fisher’s
LSD) comparisons revealed that tension decreased in TnT1–44D reconsti-
tuted fibers, whether the Tm background was TmWT or TmDM. Maximal
tension remained unaffected in TmDMþTnT45–74D fibers, but attenuated
significantly in TmH276NþTnT45–74D fibers (16), suggesting that the
S229E-induced instability in the central region of TmDM affected the over-
lapping ends of TmDM. Number of determinations was at least six in each
group. Values are reported as mean 5 SE. ***P < 0.0001.
2107
TABLE 1 Steady-state contractile parameters of TmWT and
TmDM TG cardiac muscle fibers reconstituted with the WT
mouse cardiac recombinant Tn complex
TnTWTþTnIþTnC TnTWTþTnIþTnC
reconstituted into reconstituted into
TmWT cardiac TmDM cardiac
muscle fibers muscle fibers
Tension (mN$mm2) 49.75 5 0.94 48.41 5 1.33
ATPase (pmol$mm3$s1) 304.38 5 9.36 238.40 5 9.71***
Tension cost 6.39 5 0.18 4.78 5 0.23***
(pmol$mN1$mm1$s1)
pCa50-tension 5.65 5 0.01 5.58 5 0.01**
nH 2.62 5 0.13 2.74 5 0.08
ktr (s1) 12.87 5 0.46 11.98 5 0.98
Isometric steady-state force and ATPase activity were measured simulta-
neously as described previously at 20 C (25,26). The resting SL was
2.3 mm. Tension is expressed as the force per cross-sectional area. Tension
cost was estimated from the slope of the relationship between steady-state
isometric tension and ATPase activity as described previously (24). Data
from pCa-tension relationships were fitted to the Hill equation to derive
pCa50 and nH values. ktr measurements in maximally-activated (pCa 4.3)
muscle fibers was according to the modified protocol described by
Brenner and Eisenberg (30). Number of determinations was at least six
for each group. Values are reported as mean 5 SE. **P < 0.01; ***P <
0.0001.
stituted fibers, respectively. In the presence of TmDM,
values of ATPase activity were as follows: 238.40 5 9.71,
230.86 5 15.42, and 260.41 5 10.51 for TnTWT, TnT1–44D,
and TnT45–74D reconstituted fibers, respectively. Two-way
ANOVA revealed a significant interaction effect (P <
0.01), suggesting that TmDM modified the impact of
TnT1–44D and TnT45–74D on maximal ATPase activity. Post
hoc multiple comparison tests revealed that TnT1–44D—
but not TnT45–74D—had a differential impact on maximal
ATPase activity. As demonstrated in tension data, maxi-
mal ATPase activity decreased significantly by ~26% in
TmWTþTnT1–44D fibers. However, in contrast to tension
data, maximal ATPase activity remained unaltered in
TmDMþTnT1–44D fibers, suggesting that S229E ablated
the negative impact of TnT1–44D on maximal ATPase activ-
ity in TmDMþTnT1–44D fibers. Similar to tension data,
TmDM had no effect on Ca2þ-activated maximal ATPase
activity in TnT45–74D reconstituted fibers. TmDM attenuated
Ca2þ-activated maximal ATPase activity in the presence of
TnTWT (Table 1).
Effect of TmDM on the rate constant of XB
detachment (g) in TnT1–44D or TnT45–74D
reconstituted cardiac muscle fibers
The rate of ventricular relaxation decreased significantly in
TmDM TG mouse hearts (18). We measured the impact of
TmDM on tension cost to test if the attenuation of ventricular
relaxation resulted from slowing of the rate of XB detach-
ment (g). Tension cost was estimated from simultaneous
measurements of steady-state isometric force and ATPase
Biophysical Journal 105(9) 2104–2113
2108
activity (25,27). The slope of the ATPase-tension plot
(Fig. 4, A and B), is a measure of the amount of ATP
consumed for a given amount of tension (tension cost
(Fig. 4 C)). Because the ratio of ATPase (fg/fþg) to tension
(f/fþg) is proportional to g, tension cost is an approximation
of the rate constant of XB detachment, g (31,32). Two-way
ANOVA revealed a significant interaction effect (P < 0.05),
suggesting that TmDM modified the effect of TnT1–44D and
TnT45–74D on tension cost.
Post hoc multiple pairwise comparisons revealed that
TmWT and TmDM had divergent impacts on TnT1–44D- or
TnT45–74D-mediated effect on tension cost. In the presence
of TmWT, tension cost remained unaltered in TnT1–44D or
TnT45–74D reconstituted fibers (Fig. 4, A and C). However,
in the presence of TmDM, tension cost increased signifi-
cantly in TnT1–44D or TnT45–74D reconstituted fibers
(Fig. 4, B and C); specifically, tension cost increased signif-
icantly by ~29% and ~16% in TmDMþTnT1–44D fibers and
TmDMþTnT45–74D fibers, respectively, when compared to
TmDMþTnTWT fibers. Because changes in tension cost
reflect changes in g, TmDM increased g in TnT1–44D or
TnT45–74D reconstituted fibers. Interestingly, tension cost
decreased significantly in TmDMþTnTWT fibers when
compared to TmWTþTnTWT fibers (Table 1), suggesting
that TmDM decreased g in the presence of TnTWT. Thus,
the interplay between TmDM and TnT1–44D or TnT45–74D
had an opposite effect on g.
Effect of TmDM on the rate of force redevelopment
(ktr) in TnT1–44D or TnT45–74D reconstituted cardiac
muscle fibers
A novel, to our knowledge, finding in this study is that
TmDM decreased g (Table 1) in the presence of TnTWT
Biophysical Journal 105(9) 2104–2113
Mamidi et al.
but increased g in the presence of TnT1–44D or TnT45–74D
(Fig. 4, B and C). Because the XB turnover rate (ktr) is pro-
portional to the sum of f and g (31), we sought to determine
if TmDM-induced changes in XB detachment kinetics had
any effect on ktr. The effect of TmDM on Ca2þ-activated
maximal ktr was measured in muscle fibers reconstituted
with TnT1–44D or TnT45–74D. Two-way ANOVA showed
no significant interaction effect, but revealed a significant
main effect of TnT (P < 0.05) on ktr.
Post hoc multiple pairwise comparisons revealed that
TmWT did not alter ktr in either TnT1–44D or TnT45–74D
reconstituted fibers (Fig. 5). On the other hand, S229E
increased ktr in TmDMþTnT45–74D fibers (Fig. 5, A and C).
Because ktr decreases significantly in TmH276NþTnT1–44D fi-
bers (16), we believe that the S229E-mediated effects coun-
teracts the attenuating effect of H276N such that the ktr of
TmDMþTnT1–44D fibers now becomes statistically insignifi-
cant when compared to TmDMþTnTWT fibers (Fig. 5 C).
Effect of TmDM on myofilament Ca2D sensitivity
(pCa50) and cooperativity of tension development
(nH) in TnT1–44D or TnT45–74D reconstituted cardiac
muscle fibers
Because the S229E substitution in TmDM altered ktr, we
wanted to test if TmDM affected myofilament Ca2þ sensi-
tivity. Normalized tension values were plotted against a
range of pCa to estimate the pCa-tension relationships in re-
constituted fibers (Fig. 6). To estimate pCa50 values, normal-
ized pCa-tension data were fitted to the Hill equation
(Fig. 7). As demonstrated by a leftward shift of the pCa-ten-
sion relationship, TnT45–74D increased myofilament Ca2þ
sensitivity significantly in the presence of TmWT (Fig. 6
A) or TmDM (Fig. 6 B). On the other hand, TnT1–44D
FIGURE 4 Effect of TmWT and TmDM on ten-
sion cost in TnT1–44D or TnT45–74D reconstituted
cardiac muscle fibers. (A) Effect of TmWT on the
tension-ATPase relationship in TnT1–44D or
TnT45–74D reconstituted fibers. (B) Effect of
TmDM on the tension-ATPase relationship in
TnT1–44D or TnT45–74D reconstituted fibers. The
relationship between steady-state isometric tension
and the corresponding ATPase activity was deter-
mined at various levels of Ca2þ activation
(26,27). (C) Tension cost was derived from the
slope of the tension-ATPase plot. Two-way
ANOVA revealed a significant Tm-TnT interaction
effect (P < 0.05), indicating that TmDM altered the
impact of TnT1–44D and TnT45–74D on tension cost.
Post hoc multiple pairwise (Fisher’s LSD) compar-
isons revealed that tension cost remained unaf-
fected by TmWT in either TnT1–44D or TnT45–74D
reconstituted fibers but increased significantly by
TmDM in TnT1–44D or TnT45–74D reconstituted fi-
bers. Number of determinations was at least six
for each group. Values are reported as mean 5
SE. *P < 0.05; ***P < 0.001.
S229E Affects the Overlapping Ends of Tropomyosin
FIGURE 5 Effect of TmWT and TmDM on the rate of force redevelopment
(ktr) in TnT1–44D or TnT45–74D reconstituted cardiac muscle fibers. ktr was
measured as described previously (30). The resting SL was 2.3 mm and
the temperature was 20 C. (A) Effect of TmDM on tension redevelopment
following a rapid slack and restretch protocol in TnT1–44D or TnT45–74D re-
constituted fibers. Data in panel A is shown as normalized tension and traces
shown are representative samples from each group. Tension redevelopment
was fitted to a single exponential to derive the rate constant ktr. (B) Repre-
sentative length trace used in the slack/restretch protocol for ktr estimation.
(C) Effect of TmWT and TmDM on ktr in TnT1–44D or TnT45–74D reconstituted
fibers. Two-way ANOVA revealed no significant interaction effect (P ¼
0.08) on ktr. Post hoc multiple pairwise comparisons revealed that TmWT
did not alter ktr in either TnT1–44D or TnT45–74D reconstituted fibers. As
shown in panels A and C, TmDM increased ktr in TnT45–74D reconstituted fi-
bers but not in TnT1–44D reconstituted fibers. Number of determinations was
at least six in each group. Values are reported as mean 5 SE. **P < 0.01.
attenuated myofilament Ca2þ sensitivity significantly in the
presence of TmWT as indicated by a rightward shift of the
pCa-tension relationship (Fig. 6 A). TmDM had a significant
effect on the pCa-tension relationship in TnT1–44D reconsti-
tuted fibers because the rightward shift was ablated in TmDM
þTnT1–44D fibers (indicated by arrows in Fig. 6 B).
Two-way ANOVA revealed a significant interaction
effect (P < 0.05), suggesting that TmWT and TmDM had a
divergent impact on how TnT1–44D and TnT45–74D altered
pCa50 (Fig. 7 A). Post hoc multiple pairwise comparisons
showed that pCa50 decreased significantly in TmWT
þTnT1–44D fibers; however, pCa50 increased significantly
in TmWTþTnT45–74D and TmDMþTnT45–74D fibers (Fig. 7
A). Despite the lack of any difference in pCa50 between
TmDMþTnTWT and TmDMþTnT1–44D fibers (Fig. 7 A),
2109
FIGURE 6 Effect of TmWT and TmDM on the pCa-tension relationships
in TnT1–44D or TnT45–74D reconstituted cardiac muscle fibers. Normalized
pCa-tension data were fitted to the Hill equation. (A) Effect of TmWT
on the pCa-tension relationship in TnT1–44D or TnT45–74D reconstituted
fibers. (B) Effect of TmDM on the pCa-tension relationship in TnT1–44D
or TnT45–74D reconstituted fibers. Arrows in panel B indicate that
the S229E substitution in TmDM attenuates the desensitizing effect of
TnT1–44D on myofilament Ca2þ sensitivity (see TmWTþTnT1–44D fibers
in panel A), thereby ablating the TnT1–44D-induced decrease in myofilament
Ca2þ sensitivity. Number of determinations was at least six in each group.
Values are reported as mean 5 SE.
this particular observation reveals the significant effect of
the S229E substitution on myofilament Ca2þ sensitivity.
TnT1–44DþTmH276N decrease myofilament Ca2þ sensitivity
significantly (16), but this desensitizing effect on myofila-
ment Ca2þ sensitivity is ablated by S229E; for example,
there is no statistical difference between TmDMþTnTWT
and TmDMþTnT1–44D fibers (Fig. 7 A). Normalized pCa-
tension data were fitted to Hill’s equation to estimate nH
values. Two-way ANOVA showed no significant interaction
effect, but revealed a significant main effect (P < 0.0001) of
TnT1–44D and TnT45–74D on nH (Fig. 7 B). Post hoc multiple
pairwise comparisons revealed that TnT45–74D significantly
decreased (P < 0.01) nH by ~27% and ~23% under TmWT
and TmDM backgrounds, respectively (Fig. 7 B). On the
other hand, TnT1–44D did not affect nH, regardless of the
type of Tm present.
DISCUSSION
The data presented in this study show that subtle structural
instability in the central region of Tm modulates the activity
Biophysical Journal 105(9) 2104–2113
2110
FIGURE 7 Effect of TmDM on myofilament Ca2þ-sensitivity (pCa50) or
cooperativity of tension development (nH) in TnT1–44D and TnT45–74D re-
constituted cardiac muscle fibers. pCa-tension data were fitted to Hill’s
equation to derive pCa50 and nH values. (A) Effect of TmWT and TmDM
on pCa50 in TnT1–44D or TnT45–74D reconstituted fibers. Two-way
ANOVA revealed a significant Tm-cTnT interaction effect (P < 0.05), indi-
cating that TmDM altered the impact of TnT1–44D and TnT45–74D on pCa50.
Post hoc multiple pairwise comparisons revealed that pCa50 decreased
significantly in TmWTþTnT1–44D fibers, but not in TmDMþTnT1–44D fibers.
TnT45–74D significantly increased pCa50 in the presence of TmWT or TmDM.
(B) Effect of TmWT and TmDM on nH in TnT1–44D or TnT45–74D reconsti-
tuted fibers. Two-way ANOVA revealed no significant Tm-cTnT interaction
effect (P ¼ 0.70) on nH. Post hoc multiple pairwise comparisons revealed
that nH decreased significantly by TnT45–74D in the presence of TmWT or
TmDM. Number of determinations was at least six in each group. Values
are reported as mean 5 SE. **P < 0.01; ***P < 0.001.
of overlapping ends of contiguous Tm dimers. We used a
TG mouse model (TmDM) that expressed a mutant a-Tm
with S229E and H276N substitutions; these substitutions
induce structural instability in the central region and the
overlapping ends of Tm, respectively (16,18). To our know-
ledge, novel findings in this study show that the S229E
substitution modulates the H276N-mediated effect on the
overlapping ends of TmDM. We discuss these results in
terms of new, to our knowledge, evidence for the emerging
paradigm of disparate Tm functions facilitated by intrinsic
structural instability in the central region of Tm.
The S229E mutation-mediated structural
instability in Tm affects cardiac contractile
function
S229 lies in the central region (residues 143–235) of a-Tm
that is known to be intrinsically unstable (Paulucci et al.
(14); and schematically represented in Fig. 1). We have
also previously shown that the S229E-induced charge alter-
ations produce structural instability in Tm to bring about
altered contractile function. Electrostatic charge analysis
Biophysical Journal 105(9) 2104–2113
Mamidi et al.
and surface rendering of Tm demonstrates that S229E
disrupts Tm-Tm contacts by modifying the local electro-
static potential of the surface of Tm and by increasing the
distance between the Ca carbon atoms and carbonyl atoms
at residues 229–231 of the two intertwined Tm helical
strands (18). S229E not only decreases myofilament Ca2þ
sensitivity in skinned muscle fiber preparations, but also
attenuates rates of relaxation and pressure development at
the whole heart level (33). These observations clearly
show that structural instability in the central region of Tm
modulates cardiac contractile function. Such observations
are further supported by studies of cardiomyopathy-related
mutations in the central region of Tm. For example,
D230N in Tm affects contractile function by attenuating
myofilament Ca2þ sensitivity (34). D175N and E180G
mutations in Tm increase the flexibility of Tm both locally
and globally—an effect that increases Ca2þ sensitivity by
shifting the on/off state of thin filaments more toward
the on state (35). However, the mechanism by which the
structural instability in the central region of Tm modulates
contractile function has remained largely unknown.
The S229E mutation-mediated structural
instability in Tm alters Ca2D-activated maximal
tension via its effect on the overlapping ends
of Tm
An interesting observation about TmDM was that the S229E
substitution in TmDM significantly altered the H276N-medi-
ated effect on contractile function, suggesting a long-range
effect of S229E on the overlapping ends of contiguous
TmDM dimers. Ca2þ-activated tension measurements sug-
gested that the S229E substitution in TmDM significantly
altered the TnT45–74D-mediated effect on the overlapping
ends of TmDM. For example, TmH276N attenuated maximal
tension in TnT45–74D reconstituted fibers (16), but TmDM
had no effect on maximal tension in TnT45–74D reconstituted
fibers (Fig. 3). This suggested that the S229E-mediated
structural changes had a long-range conformational effect
to alter the interaction between TnT45–74D and the overlap-
ping ends of contiguous TmDM.
Previously, we demonstrated that a-Tm did not alter
the rate constant of XB detachment (g) in TnT1–44D re-
constituted fibers, but b-Tm increased g significantly in
TnT1–44D reconstituted fibers (17), suggesting that Tm iso-
forms altered the way TnT1–44D modified cardiac thin fila-
ment activation. In this study, we showed that S229E and
H276N substitutions in TmDM increased g in TnT1–44D re-
constituted fibers (Fig. 4). Given how TmH276N decreases
g (16), the augmenting effect of S229E on g becomes
apparent (Fig. 4). Interestingly, b-Tm contains two charge-
modifying amino acid substitutions (S229E and H276N)
that are different in a-Tm (18), leading us to conclude that
the S229E substitution has a long-range effect on the over-
lapping ends of adjoining b-Tm dimers.
S229E Affects the Overlapping Ends of Tropomyosin
The S229E-induced instability in the central
region of TmDM alters the rate constant of force
redevelopment (ktr)
A change in ktr could be attributed to an effect on the equi-
librium between the closed- and open-states (36,37) of car-
diac thin filaments because ktr is a measure of the rate of
transition of XBs from the weak- to strong-binding states
(30,38). An effect on the closed- to open-state transition
of thin filaments may have an impact on myofilament
Ca2þ sensitivity. Measurement of Ca2þ-activated maximal
ktr demonstrated that S229E altered ktr significantly.
Ca2þ-activated maximal ktr increased significantly in TmDM
þTnT45–74D fibers (Fig. 5), but not in TmH276NþTnT45–74D
fibers (16). This is indicative of an augmenting effect of
S229E on ktr in TmDMþTnT45–74D fibers. Furthermore,
S229E also increased ktr in TmDMþTnT1–44D fibers. At first
glance, a statistically nonsignificant difference in ktr be-
tween TmDMþTnTWT and TmDMþTnT1–44D fibers (Fig. 5)
is at odds with the assertion that S229E increases maximal
ktr in TmDMþTnT1–44D fibers. However, it must be noted
that Ca2þ-activated maximal ktr decreased significantly
in TmH276NþTnT1–44D fibers (16) but not in TmDM
þTnT1–44D fibers (Fig. 5). These observations lead us to
conclude that the augmenting effect of S229E on ktr negates
the attenuating effect of H276N on ktr in TmDMþTnT1–44D
fibers. Collectively, these results are strongly suggestive
of the S229E-mediated effect on ktr in TnT1–44D or
TnT45–74D reconstituted fibers.
The S229E-induced instability in the central
region of TmDM alters myofilament Ca2D
sensitivity
Another novel, to our knowledge, finding in this study
showed that the attenuation of myofilament Ca2þ sensi-
tivity—observed in TmWTþTnT1–44D fibers (Figs. 6 A
and 7 A) and TmH276NþTnT1–44D fibers (16)—was ablated
in TmDMþTnT1–44D fibers (Figs. 6 B and 7 A). These
observations clearly demonstrated that S229E affected
myofilament Ca2þ sensitivity—more specifically, it af-
fected Ca2þ sensitivity by altering the overlapping ends
of contiguous TmDM dimers. We propose that S229E-medi-
ated changes in Tm-Tm interactions induce a long-range
effect on the overlapping ends of Tm. Whether or not
S229E-mediated effects are transduced via Tm alone or
through Tn require further investigation. Given the obser-
vations that TnI and TnT bind near Cys190 of Tm
(20,39,40), it is possible that the S229E-mediated local
instability in the central region of Tm is transduced to
the overlapping ends of two contiguous Tm through TnT
and/or TnI. However, several recent studies point to the
possibility that the transduction of changes in Tm may
go through Tm alone. For example, studies of cardiomyop-
athy-related mutations (D175N and E180G) in the central
2111
region of Tm indicated that the affinity of Tm to Tn was
not affected by both mutations (41). This observation leads
us to suggest that the S229E mutation in Tm may not have
affected the affinity of Tm for Tn; the effects observed are
possibly due to a long-range impact of S229E on the over-
lapping region of Tm. This is further supported by recent
biochemical studies which show that D175N and E180G
mutations destabilize a large part of Tm (42). D175N and
E180G mutation-induced destabilization lead to a signifi-
cant increase in the rate of trypsin-mediated cleavage at
R133 of Tm, a site that is located ~45 residues N-terminal
to mutations in Tm (42). Furthermore, a recent NMR spec-
troscopy study shows that the D137L mutation in Tm pro-
duces long-range structural rearrangements in Tm (43).
Collectively, these results strongly support our hypothesis
that S229E has the potential to bring about long-range
structural alterations in Tm.
The attenuation of Ca2þ sensitivity by TnT1–44D—in the
presence of either TmWT or TmH276N—was previously
explained by the TnT1–44D-mediated increase in the rigidity
of Tm (17). Such an impact on Tm would shift the off/on
equilibrium of the regulatory units (RU: Tm-Tn) more
toward the off state; thereby, attenuating myofilament
Ca2þ sensitivity in TmH276NþTnT1–44D reconstituted fibers.
Similar to the observation made in ktr experiments
(Fig. 5 C), pCa50 decreased significantly in TmH276N
þTnT1–44D fibers (16)—but not in TmDMþTnT1–44D fibers
(Fig. 6 B)—indicating that the augmenting effect of
S229E on myofilament Ca2þ sensitivity negated the attenu-
ating effect of TnT1–44D on myofilament Ca2þ sensitivity in
TmDMþTnT1–44D fibers. Inferences made from the previous
analysis lead us to conclude that the S229E substitution in
TmDM affects the transition between off/on states of the
RU. For the first time (to our knowledge), these observations
provide a mechanistic basis for the b-Tm-mediated increase
in cardiac myofilament Ca2þ sensitivity observed in previ-
ous studies (22,23,44,45).
Recent studies showed that TmH276N did not alter ktr in
TnT45–74D reconstituted fibers (16). In contrast, findings
from this study showed that TmDM increased ktr in
TnT45–74D reconstituted fibers (Fig. 5), which indicated
that S229E modulated XB-mediated transition of the thin
filament from the closed- to open-states. Previously, we
showed that TnT45–74D enhanced myofilament Ca2þ sensi-
tivity by favoring the transition of the thin filament from
the blocked- to closed-states (17,46), a property that appears
to be still operative in TmH276NþTnT45–74D or TmDM
þTnT45-74D fibers. If the closed- to open-state transition of
the thin filament is augmented in TmDMþTnT45–74D fibers,
in addition to the shift in the off/on equilibrium of the RU,
we expect TmDMþTnT45–74D fibers to be more sensitive to
Ca2þ than TmH276NþTnT45–74D fibers. Indeed, that is what
we observe when the pCa50 of TmH276NþTnT45–74D (16)
and TmDMþTnT45–74D fibers (Fig. 6 B) are compared
(pCa50 of 5.93 vs. 6.03).
Biophysical Journal 105(9) 2104–2113
2112
CONCLUSION
This study provides insights into the mechanism by which
conformational plasticity in the central region of Tm
modulates cardiac contractile function. The data presented
in this study show that the structural instability in the central
region of Tm is transduced to the overlapping ends of Tm to
alter cardiac contractile function. Because subtle regional
instability does not affect the overall stability of Tm binding
to actin (10) or Tn (41), we believe that altered contractile
function results from the long-range impact on the over-
lapping ends where Tm and TnT interact. Varied expression
of Tm flexibility—caused either by disease-related substitu-
tions or naturally occurring amino acid substitutions in
tissue-specific Tm isoforms—may lead to disparate physio-
logical functions.
SUPPORTING MATERIAL
One figure, supplemental information and references (50–53) are
available at http://www.biophysj.org/biophysj/supplemental/S0006-
3495(13)01074-6.
The authors thank Sri Lakshmi Mallampalli for excellent technical assis-
tance and Dr. Sampath Kumar Gollapudi for help in preparing figures.
This work was funded by National Heart, Lung, and Blood Institute Grant
R01-HL-075643 (to M.C.), KO2-HL-86650 (to M.M.), and Poncin fellow-
ship (to R.M.). There are no conflicts of interest.
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