Dear Dr. Regnier, Dr. Neils, Dr. Taylor, and Dr.

Hendricks,
This is the final draft of my BIOEN 403 capstone research paper. I have been revising this
paper with the support of my peers over the past year. I have received feedback on lower order
concerns such as grammar, verb tense, and headers to higher order concerns like structure and
flow of the various sections. This draft looks and reads differently than the one I turned in last
month because I strove to incorporate all of the thoughtful feedback I got from my classmates. I
was told to incorporate more scientific literature around distal arthrogryposis (DA) because I had
too much information on the disease that is the cardiac counterpart of DA. I was also informed
that my previous introduction was unclear since it did not have a firm hypothesis, clear aim, or
or scientific motivation behind my study, I appreciate all the time and effort my peers have put
into supporting me throughout the writing process, and I also appreciate each of you for taking
the time to support me in my research endeavors in different ways. I hope you enjoy reading it
as much as I did writing it.
Sincerely,
Min Jung Kim

Altered regulation of thin filament sliding implicated in an In Vitro

Motility assay with mutated troponin T found in Distal Arthrogryposis
Min Jung Kim
PI: Dr. Michael Regnier
Mentor: Marta Sierra and Alice Ward Racca

ABSTRACT
Distal Arthrogryposis (DA) is an umbrella term for a group of disorders that is characterized by
non-progressive congenital skeletal muscle defect and inherited in an autosomal dominant
manner. The purpose of this study is to expand on a hypothesis that R63H mutation in troponin
T changes the conformation of thin filament regulatory complex to favor the strong myosinbinding open state of a three state steric blocking model. An in vitro motility (IVM) assay was
performed to determine that the change in single amino acid leads to changes in the functional
role of TnT in thin filament sliding in an Ca2+ independent manner that blocks the open state of
actin.

INTRODUCTION

includes subunits C, I, and T. Tn works in

conjunction with another protein called
tropomyosin (Tm) to control the thin filament
and to ultimately mediate the forcegenerating interactions between myosin and
actin. This thin filament regulatory complex
is thought to provide steric control of the
actomyosin in three states (McKillop et al.
1993). In the absence of calcium, the thin
filament regulatory complex is thought to
physically inhibit actomyosin interaction by
covering the myosin-binding pocket on actin
(blocked state). When calcium binds to Tn,
series of conformational changes in the thin
filament result in the movement of Tm on
the surface of actin to allow partial myosin
attachment to actin (closed state). Then this
complex has been suggested to isomerize
to give rise to strongly bound actomyosin
and weakly bound nucleotide (open state).
In addition to this three state steric blocking
model, the thin filament regulatory complex
is thought to also alter the kinetics of the
actomyosin interaction by modulating the
size of the power stroke (LaMadrid 2002),
the apparent nucleotide binding affinity to
actomyosin (Homsher et al. 2003), number
of cross-bridge heads formed (Homsher et
al1996), and rate at which ATP is
hydrolyzed.

Distal Arthrogryposis (DA) is an umbrella

term for a group of disorders that is
characterized by non-progressive congenital
skeletal muscle defect and inherited in an
autosomal dominant manner. Patients with
DA have lifelong abnormal contractures of
two or more different body areas without
signs of neurological disease (Bamshad et
al 1996). Some of the more common forms
of the disease include club foot,
camptodactyly whistle mouth, and
nasolabial folds (Bamshad et al. 2009). It
occurs in 1 in every 3,000 live births (Fahy
and Hall 1990). The precise molecular
mechanism behind the malformations are
largely unknown. As a result, there are no
drugs for the disease, and patients usually
go through surgery and or regular physical
therapy throughout their lifetime. Severe
cases require surgical intervention and
amputation.
Hall et al. (1982) first created the DA
classification system after summarizing a
large study of 350 patients with congenital
joint contractures and categorized the
condition into two main groups. Bamshad et
al. (1996) revised and extended the original
classification and recognized ten different
DAs. Since then, they have been performing
genetic linkage analysis to create a
chromosomal map of the disease. Of the
ten subtypes of DA, DA2B has been shown
to be caused by two mutations in TNNI2
and TNNT3 on chromosome 11p15.5. that
encode for fast skeletal isoforms of troponin
I and troponin T respectively (Sung et al.
2003a). The troponin T (TnT) mutation is a
missense mutation that results in an
arginine to histidine substitution at the 63rd
base pair (R63H). Interestingly, this arginine
residue is known to be conserved among all
isoforms of TnT (Sung et al. 2003b) and
thus thought to have an important functional
role.

This mutation is found on the N terminal

region of TnT, and this region is interesting
to study because alternative RNA splicing is
used heavily to generate a diversity of more
than 60 TnT isoforms (Breitbart et al. 1985)
(Fig. 1). Many hypothesize that the N
terminus must be responsible for fine tuning
the proteins diverse functional roles in
contractility of a cell since transcription of
this region is so tightly controlled
spatiotemporally. The 63rd amino acid is
found between the regions of the TnT that
are thought to bind to tropomyosin and the
area that is meticulously controlled.
Since 1) TnT is part of a larger thin filament
regulatory complex that is thought to have
both steric and allosteric roles in and 2) the
R63H mutation is found on a conserved
residue in an area of TnT that is highly

TnT is a part of a trimeric protein complex

called the troponin complex (Tn) that
3

Figure 1: A schematic representation of adult fast skeletal TnT molecule is reproduced above from a
paper of Perry SV (1998). Hatched areas mark the regions of the gene that is most often spliced for
producing a wide array of isoforms .
regulated, investigating the impact of R63H
TnT mutation on thin filament in DA will not
only give us great insight into thin filament
regulations role in development of limb
defects but also shed light on the functional
role of TnTs N terminal.

METHODS
Myosin and Muscle Bundle Fibers
Preparation
Rabbit fast skeletal myosin were obtained
from psoas muscle of New Zealand white
rabbits. They were euthanized with 50
mg/kg of sodium pentabarbitol in the
marginal ear vein, in accordance with NIH
animal care policy and approved by the
University of Washington Animal Care
Committee. Skeletal myosin was extracted
using Margossian and Loweys methods
(1982) and stored at 20C in a high
phosphate solution (0.5 mM KCl, 10 mM
NaHPO4 , 2 mM MgCl2 , 1 mM DTT) with 50%
glycerol. The day before an IVM experiment,
tosyl-lysine chloromethyl-ketone (TLCK)
chymotrypsin (Sigma-Aldrich, St. Louis, MO)
were used to digest stock myosin aliquots
into yield heavy meromyosin (HMM) (Sung
et al. 2003b). For each day of IVM, weekly
HMM were centrifuged with actin to remove
denatured HMM as previously described
(Margossian and Lowey 1982).
Spectrophotometry was used to determine
HMM and myosin concentrations as
previously described (Clemmens and
Regnier 2004).

Previously, Robinson et al. (2008) used the

actomyosin ATPase assay and troponin
replaced rabbit psoas fibers to study the
impact of R63H on skeletal muscle
contractility. They reported that the R63H
mutation moderately increased ATPase
activity throughout the pCa range and also
increased the Ca2+ sensitivity of force
generation. However, the maximum Ca2+
activated force produced by fibers was not
significantly different. The purpose of this
study is to expand on these findings from
Robinson et al. and investigate the effect of
the R63H mutation on speed of unloaded
and regulated thin filaments (Vf). An In vitro
motility (IVM) assay was used to determine
that the change in single amino acid leads
to changes in the functional role of TnT in
thin filament sliding speed in an Ca2+
independent manner that blocks the open
state of actin.

Actin Preparation

Assay chambers were made with two

coverslips. One coverslip with dimensions of
22 x 60 mm (VWR Scientific, West Chester,
PA) was coated with 0.1% nitrocellulose in
amyl acetate (Sigma, St. Louis, MO). Two
narrow foam spacers were cut out and
placed on the dried glass to create the
chamber. One 18 mm2 coverslip was placed
on top of the foam spacers. HMM (200-300
g/mL) was injected into the chamber and
incubated for 3 minutes. This was followed
by 150 L of 0.5 mg/mL of bovine serum
albumin (Sigma, St. Louis, MO) and
incubated for 1 min to prevent unspecific
binding. The slide chamber was then
washed with 200 L of assay buffer
containing 20 mM KCl, 10 mM 3-(Nmorpholino) propanesulfonic acid (MOPS)
(pH 7.4 at 250C), 2 mM MgCl2, 1 mM DTT.
Next, shredded actin filaments were washed
into the chamber and incubated for a minute
and then washed with a solution of 2 mM
ATP. The active HMM released the
shredded actin in the presence of ATP, and
this step acted to block any dysfunctional
HMM heads. Then 150 L of the RP-labeled
thin filaments was introduced into the
chamber and incubated for 1 min. Finally,
the chamber was washed with a solution
containing regulatory proteins (tropomyosin,
troponin complex) and incubated for 6
minutes. This was followed by the activating
solution with regulatory proteins, ATP, and
calcium. All activation solutions contained 3
mg/mL glucose, 100 g/mL glucose oxidase,
10 ug/mL catalase, and 10 mM DTT to
minimize photo-bleaching of the fluorescent
label.

Rabbit skeletal F-actin were prepared from

acetone powder, labeled with rhodamine
phalloidin (RP) (Molecular Probes, Eugene,
OR) as previously described, and stored at
4C, and used within 6 weeks [9]. The
psoas fibers were placed in skinning
solution (50% glycerolrelaxing (v/v)
solution with 1% Triton detergent and 1:200
(v/v) Protease Inhibitor Cocktail (SigmaAldrich, P8340) and incubated at 4C for 2
4 hrs. The solution was then changed to a
50% glycerolrelaxing solution without
detergent and stored in at 4C.
Reconstituting the Human Troponin
Complexes
Three types of human troponin complexes
were expressed in B21 E. coli and purified
via column chromatography and frozen: wild
type (WT), WT with flag, and R63H with flag.
The frozen recombinant troponin I, T, and C
proteins were separately dialyzed in 6 M
urea buffer using Silde-A-Lyzer Dialysis
Cassettes (Life Technologies, Carlsbad,
CA). The concentration of each protein was
determined with Bradford Protein Assay
(Bio-Rad Laboratories, Redmond, WA)
using a Bovine Serum Albumin standard
curve in a linear range of 0.125 to 1.5
mg/mL and spectrophotometrically ( = 280
nm; extinction coefficient of TnC, TnI, and
TnT used were 0.247, 0.477, and 0.449 cm1
respectively). Purity of proteins were
verified by SDS-gel electrophoresis. Two
complexes, one wild type (WT) and one with
mutated TnT (R63H) were reconstituted
from the recombinant proteins and CaCl2
was added to make the final concentration
1.5 mM. The reconstituted complex was left
at room temperature for 30 to 60 minutes.
The complex was gradually dialyzed using
the cassette mentioned above to a final
working solution consisting of 150 mM KCl,
25 mM imidazole, 2 mM MgCl2, 1 mM EGTA,
and 1 mM DTT. The final pH was brought to
7.

Data Acquisition and Analysis

Videos of photo-labeled actin were acquired
using IVM Data Acquisition and IVM Data
Analysis Programs that were developed by
Martha Mathiason, an IT specialist in Heart
and Muscle Mechanics Lab. The recordings
were typically 10 seconds long. Brightness
and contrast was adjusted per each
recording to yield the best video resolution
by eye. Each pixel had an area of 1 m2.

In vitro motility assays

5

Vf (m/s)
[Tn] (nM)
170
200

WT
6.31 0.11
6.27 0.23

R63H
6.49 0.10
6.54 0.10

Fraction moving (%)

WT
R63H
6.63 0.05
6.96 0.09
6.37 0.19
6.54 0.10

Table 1: pCa50 from 4 parameter Hill equation fit found in SigmaPlot of thin filaments regulated with
WT and R63H Tn are summarized above.

When the acquired video files were

analyzed, the program identified each
filament as an object and the objects were
analyzed only if they were between 3 400
pixels big and identifiable in the video for
more than 15 frames. A threshold of 10
pixels per frame was set as the maximum
filament speed. Filaments were determined
to be moving if they 1) not move during part
of the recording, 2) cross paths with another
filament, 3) get tangled with their own tail, or
4) get pulled apart into separate pieces.
Mean and standard deviation of filament
speed for the duration of the video recording
was determined for each filament that met
these criteria as described previously in the
literature (Homsher 1992; Clemmens and
Regnier 2004). If the ratio of standard
deviation to mean speed calculated was no
more than 1 to 1, the filament was
determined to be non-erratically moving.
200 - 800 filaments were analyzed for each
calcium concentration with the same Tn
complex. Mean and standard deviation of
moving thin filament velocity and fraction
were plotted using Sigma Plot. 4 parameter
Hill equation was used as non linear
regression:
# =

1+

RESULTS
To study the effect of R63H TnT mutation
on unloaded thin filament sliding, an in vitro
motility assay was performed with
reconstituted Tn from recombinant troponin
subunits that were expressed, extracted,
and purified from E. coli.
Effect of mutation on velocity and
fraction of thin filaments moving
Thin filament regulation with R63H TnT was
found to have statistically significant impact
on increasing thin filament sliding velocity at
all calcium concentrations tested (9, 8, 7,
6.8, 6.6, 6.4, 6.2, 6, 5.8, 5.5, and 5)
regardless of regulatory protein
concentration (Fig. 1). The only exception
for this observation was found at saturating
Ca2+ concentration (pCa = 5.0) with
regulatory protein concentrations of 170 nM.
Ca2+ is thought to control the total number of
cross-bridges interacting with thin filament
to indirectly modulate thin filament sliding
speed (Gordon et al. 2000). Our finding that
thin filament speed is generally greater with
R63H Tn compared to WT Tn implies that
the mutation modulates a Ca2+ independent
functional role in increasing thin filament
sliding speed.

%&'
1
,-(/0&1/0&23)
10

pCa50 is the pCa value that yields half

maximum thin filament velocity. This
parameter can be used to compare the
calcium sensitivity of thin filament velocity
under various conditions. nH is the Hill
coefficient. It is a measure of steepness of
the curved portion of a sigmoidal graph and
it also gives insight into apparent
cooperativity of the thin filament activation.

Effect of mutation on thin filament

sliding cooperativity
With the exception of fraction of moving
filaments with 170 nM, Tn R63H (Fig. 3A),
Mutated Tn is shown to have more
cooperative regulation of thin filament
sliding compared to WT Tn. This is
illustrated by the steeper slopes of the Hill fit
curves for R63H Tn compared to WT Tn
6

Figure 2: The effect of Tn regulation with WT Tn (open dot) and R63H (closed dot) on sliding
speed of thin filament is shown above. Each point represents the mean and standard deviation
of filament speeds at pCa of 9, 8, 7, 6.8, 6.6, 6.4, 6.2, 6, 5.8, 5.5, and 5. Two concentrations of
regulatory proteins were tested: 170 nM (A) and 200 nM (B). The solid and dotted lines in each
plot are 4 parameter Hill equation fits with Tn with R63H and WT complexes respectively. With
170 nM of regulatory proteins, the pCa50 of R63H and WT Tn were 6.49 m/s and 6.31 m/s
respectively. With 200 nM of regulatory proteins, the pCa50 of R63H and WT Tn were 6.54
m/s and 6.27 m/s respectively.
7

Figure 3: The effect of regulatory proteins WT Tn (open dot) and R63H (closed dot) on fraction
of non erratically moving filaments were determined in vitro. Each point represents the mean
and standard deviation of filament speeds at pCa of 9, 8, 7, 6.8, 6.6, 6.4, 6.2, 6, 5.8, 5.5, and 5.
Two concentrations of regulatory proteins were tested: 170 nM (A) and 200 nM (B). The solid
and dotted lines in each plot are 4 parameter Hill equation fits with Tn with R63H and WT
complexes respectively. With 170 nM of regulatory proteins, the pCa50 of R63H and WT Tn
were 6.96 m/s and 6.63 m/s respectively. With 200 nM of regulatory proteins, the pCa50 of
R63H and WT Tn were 6.54 m/s and 6.37 m/s respectively.
8

(Fig. 2 and 3). Curved fits for fraction of thin

filaments moving was steeper that that of Vf,
which implies that the mechanisms that
control the transition steps between rigor
and moving thin filaments work in a more
cooperative manner compared to those of
thin filament speed. This was an expected
observation. Previously, studies have
shown that speed is not very cooperative
while the number of sliding filaments
generally exhibit greater cooperativity
(Gordon et al. 2000).

speed (Gordon et al. 2007; Homsher et al.

1996; Homsher et al. 2000; Lin et al. 1996).
Both of these conclusions indicate that thin
filament regulatory proteins control muscle
contraction by modulating the number of
attached and cycling cross-bridges and not
the rate or size of cross-bridge throw. As a
result, conditions that lead to change the
magnitude of speed-force pCa50 would
suggest there is a change in number of
attached and cycling cross-bridges formed
with the new condition.

Effect of mutation on Ca2+ sensitivity of

thin filament sliding

For our specific case, Robinson et al. found

that the pCa50 of force-pCa curve for R63H
Tn was 5.5. This means that speed-force
pCa50 for R63H Tn is 0.8 1.0, which is
smaller than that of WT Tn (1 1.2)
calculated earlier. Applying the argument
outlined previously and by comparing the
change in speed-force pCa50 with R63H
Tn, our data suggest that R63H Tn
promotes a smaller number of attached and
cycling cross-bridges.

R63H TnT did not have a statistically

significant impact on calcium sensitivity of
thin filament sliding velocity (Table 1). pCa50
was generally found to be around 6.3 6.5
regardless of the conditions. Our finding is
qualitatively consistent with previous studies.
pCa50 of force-pCa curves have been
shown to be 0.4 0.8 pCa units smaller
than that of speed-pCa curves (Gordon et al.
1997, Homsher et al. 1997):

This finding is consistent with the earlier

finding from comparing thin filament
velocities over a wide range of pCa values.
It has been shown that strongly attached
cycling cross bridges can delay relaxation in
skeletal muscle (Gordon et al. 2000). If the
inverse is true, smaller number of attached
and cycling cross-bridges would lead to
faster filaments. This implies that since
regulation of R63H Tn results in smaller
number of cycling cross-bridges, unloaded
sliding filaments would have greater velocity
when regulated with R63H Tn. This is the
same conclusion we have made earlier.

pCa23 of speed pCa curve

pCa23 of force pCa curve
= speed force pCa23 =
0.4 0.8
The pCa50 of force-pCa curve from
Robinson et al. was 5.3 for WT Tn. As a
result, speed-force pCa50 of WT Tn is 1
1.2. This means that our speed-pCa curve
pCa50 is slightly lower than previously
shown, but our result is qualitatively similar
to previous work in that the pCa50 of forcepCa curve is still smaller than that of speedpCa curve.

Although our findings are internally

consistent, they are not sound with the
overall conclusion made by Robinson et al.
If Tm is very flexible, one regulatory unit
(composed of 7 actin monomers, 1 Tm, and
1 Tn complex) would have one or two actins
in the open state and the rest in the closed
state with Ca2+ (Gordon et al. 2000). Two or
more actins would be in an open state with
the conclusion from the study of Robinson
et al (2007). This would in return result in a

The conclusion from previous studies that

pCa50 of force-pCa curves are smaller than
that of speed-pCa curves suggests that
observable changes in force generation
precedes changes in thin filament speed.
Additionally, other groups have concluded
that few cross-bridges are necessary to
move filaments but far more cross-bridges
are needed for filaments to reach maximum
9

larger number of attached and cycling cross

bridges and thus slow down the thin
filament sliding velocity. This conclusion
contrasts with our finding that decreased
number of cross-bridges would be formed
with the mutant thin filament regulatory
complex. Our inconsistent findings suggest
that there may be missing steps between
the two myosin binding states in the three
state steric blocking model. Although this
model is elegant and widely accepted, some
groups have suggested that it is too
simplistic (Gordon et al. 2000).

Additional studies should be carried out to

investigate if states between the two
myosin-binding states in the three state
steric blocking model exist and whether
these states can bridge the gaps in our
knowledge to help us explain the
inconsistencies between our conclusion and
findings from the paper of Robinson et al
(2007). Furthermore, experiments can be
performed to determine the relationship
between actomyosin ATPase activity, preforce to force development, thin filament
sliding, and cross-bridge kinetics and how
R63H TnT impacts those relationships.

DISCUSSION
The data presented in this report suggest
the following. TnT R63H:

In this study and many others, TnT

mutations have been associated with
changes in actomyosin ATPase, unloaded
thin filament sliding speed, or isometric
force. All of these studies suggest TnT
directly impacts a transition between
strongly attached cross-bridge states and
argue for allosteric effects on the thin
filament. With this in mind, although steric
and kinetic impacts of mutations are often
separated into fundamentally different types
models, it is likely that there are
relationships between allosteric and steric
regulation that explain some of the
inconsistencies in each of the realms of
models.

1) increases unloaded thin filament

sliding speed in a Ca2+ independent
manner
2) enhances cooperativity of thin
filaments regulatory protein complex
that controls the transition rates and
equilibriums between the general
on and off states for thin filament
sliding
3) promotes smaller number of
attached and cycling cross-bridges
through steric and/or allosteric
control

10

ACKNOWLEDGEMENTS

Regulation of contraction in striated muscle.

Physiological Reviews. 80 Apr:80(2)853
924.

I appreciate many months of excellent

technical support I have received from
Galina Flint, MD, Dr. An-Yue Tu, Martha
Mathiason, Dr. Yuanhua Cheng, and Dr.
Maria Razumova. I am also grateful for
everyone in the Heart and Muscle
Mechanics Lab for providing an
encouraging work environment.

Hall JG, Reed SD, Greene G. The distal

arthrogryposis: delineation of new entities
review and nosologic discussion. American
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Homsher E, Wang F, Sellers JR. Factors
affecting movement of F-actin filaments
propelled by skeletal muscle heavy
meromyosin. The American Journal of
Physiology. 1992 Mar;262(3 Pt 1):C714-23.

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