doi:10.1016/j.bpj.2008.12.1140


232a Monday, March 2, 2009
provide valuable insights into its mode of action. The molecular framework of
DFBP resembles that of levosimendan, thus it was chosen to mimic levosimen-
dan to establish how the cTnC-cTnI binding equilibrium is modulated. We have
utilized 2D {1H, 15N} HSQC and 2D {1H, 13C} HSQC NMR spectroscopy to
examine the binding of DFBP to cNTnC�Ca2þ in the absence and presence of
cTnI144-163 and of cTnI144-163 to cNTnC�Ca2þ in the absence and presence
of DFBP. The results show that DFBP and cTnI144-163 bind cNTnC�Ca2þ
concurrently and the affinity of DFBP for cNTnC�Ca2þ is increased ~5-10-
fold by cTnI144-163. We are in the process of determining the NMR solution
structure of cNTnC�Ca2þ�cTnI144-163�DFBP. This structure will contribute
to the understanding of the mechanism of action of levosimendan in the therapy
of heart disease. It will also provide a structural basis for the design of Ca2þ-
sensitizing drugs in general.

1192-Pos Board B36
Decreased Fatigue Tolerance In Diaphragm Muscle Of Slow Troponin T
Knockdown Mice
Hanzhong Feng, Bin Wei, J.-P. Jin.
Northshore University Healthsystem and Northwestern University Feinberg
School of Medicine, Evanston, IL, USA.
The loss of slow skeletal muscle troponin T (TnT) results in a severe type of
nemaline myopathy in the Amish (ANM). The genes encoding TnT and tropo-
nin I (TnI) are closely linked in pairs in which the 50-enhancer region of the
slow TnT gene overlaps with the cardiac TnI gene. In a mouse line with the en-
tire cardiac TnI gene deleted, a partial destruction of the slow TnT gene pro-
moter produces a knockdown effect. By crossing with transgenic mouse lines
that over-express a core structure of cardiac TnI (cTnI-ND) under the control
of cloned alpha-MHC promoter, we rescued the postnatal lethality of the car-
diac TnI gene-deleted mice with no detrimental cardiac phenotypes or leaking
expression in non-cardiac tissues. The double transgenic mice exhibited de-
creased expression of slow TnT mRNA and protein in adult diaphragm muscle.
Functional analysis of isolated muscle strips showed that the slow TnT deficient
(sTnT-KD) diaphragm had significantly decreased fatigue tolerance evident by
the faster decrease in force and slower rate of recovery as compared with that in
wild type controls. As a consequence of slow TnT deficiency, the sTnT-KD di-
aphragm muscle contained a higher proportion of fast TnT, decreased slow TnI
with increased fast TnI, and decreased type I myosin with increased type II my-
osin. Consistent with the switch toward fast myofilament contents, the sTnT-
KD diaphragm muscle produced higher specific tension in twitch and tetanic
contractions as well as shorter time to develop peak tension in twitch contrac-
tions. The decreased fatigue tolerance of sTnT-KD diaphragm muscle explains
the terminal respiratory failure seen in virtually all ANM patients and this dou-
ble transgenic mouse model provides a useful experimental system to study the
pathogenesis and treatment of ANM.

1193-Pos Board B37
Troponin Isoforms and Stretch-activation of Insect Flight Muscle
Uros Krzic1, Gian De Nicola2, Vladimir Rybin1, Annalisa Pastore2,
Kevin Leonard3, Wolfgang Linke4, Belinda Bullard5.
1EMBL, Heidelberg, Germany, 2NIMR, London, United Kingdom, 3EBI,
Cambridge, United Kingdom, 4University of Muenster, Muenster, Germany,
5Department of Biology, University of York, United Kingdom.
Oscillatory contraction of insect indirect flight muscle (IFM) is activated by si-
nusoidal length changes. Work done by oscillating fibres is measured from the
area of loops on a length-tension plot. At [Ca2þ] above 10 mM, progressively
less oscillatory work is produced because fibres contract isometrically and are
unable to relax fully after each cycle of oscillation. Periodic stretches during
oscillations activate fibres through the action of TnC F1, which binds one
Ca2þ in the C-lobe. Activation of isometric contraction by Ca2þ acts through
F2, which binds Ca2þ in both N- and C-lobes. Lethocerus IFM fibres
substituted with F1gave oscillatory work, which did not decline at high
[Ca2þ], while fibres substituted with F2 produced more isometric tension as
[Ca2þ] was increased. Varying proportions of F1 and F2 gave maximal
work with an F1:F2 ratio of 100:1, which is higher than the in vivo ratio of
7:1. The structure of F1, and the interaction with TnI, were determined by
NMR. The N-lobe of F1 is in the closed conformation in apo and Ca2þ- bound
forms and does not bind TnI. Unexpectedly, the C-lobe is open in both states,
and binds the N-terminal domain of TnI independently of Ca2þ. The affinity of
F1and F2 for a complex containing tropomyosin, TnT and TnH (Lethocerus
TnI) were measured by isothermal calorimetry in the presence of Ca2þ. The
affinities of F1 and F2 for the complex were 5.4 mm and 65 nM respectively.
This difference is likely to be due to a single TnI binding site on F1 and two
sites on F2. Stretch may be sensed by an extended C-terminal domain of
TnH, and transmitted to the C-lobe of F1, resulting in a change in the interac-
tion of the TnI inhibitory domain and actin.
1194-Pos Board B38
Tracking of Qdot Conjugated Titin Antibodies in Single Myofibril Stretch
Experiments Reveals Ig-domain Unfolding at Physiological Sarcomere
Lengths
Anika Grützner1, Pallav Kosuri2, Julio M. Fernandez2,
Wolfgang A. Linke1.
1University of Münster, Münster, Germany, 2Columbia University,
New York, NY, USA.
The mechanical characteristics of titin in muscle sarcomeres were previously
studied by us in single myofibril stretch experiments, where the extensibility
of I-band titin segments was usually measured under static conditions. Here
we investigated the behavior of I-band titin during and after stretch of single
rabbit psoas myofibrils in real-time. The focus was on titin’s proximal Ig-do-
main region, whose stretch dynamics were analyzed by labeling the myofibrils
specifically in the N2A-titin domain using antibody-conjugated quantum dots,
which stained the periphery of the myofibril but did not enter the myofilament
lattice. Qdot labels were tracked to obtain the stretch-dependent change in epi-
tope distance (across Z-disc) and sarcomere length (SL) over time. In contrast
to what was expected from the current titin extensibility model, at sarcomere
lengths of 2.5 and 3.8 mm, titin’s proximal Ig-domain region elongated contin-
uously, in proportion to the half I-band length. Already at ~2.6 mm SL the prox-
imal Ig-segment length exceeded the value expected if all Ig-domains remain
folded. Our results suggest that Ig-domains unfold in parallel with PEVK-titin
extension at physiological sarcomere lengths and under relatively low forces.
By reducing the antibody-Qdot concentration, we succeeded in observing titin
Ig-domain dynamics in myofibrils at the single-molecule level.

1195-Pos Board B39
Constitutive Phosphorylation of Cardiac Myosin Binding Protein-C
Increases the Probability of Myosin Cross-bridge Interaction with Actin
Brett A. Colson1, Tanya Bekyarova2, Matthew R. Locher1, Carl W. Tong1,
Daniel P. Fitzsimons1, Patricia A. Powers1, Thomas C. Irving2,
Richard L. Moss1.
1University of Wisconsin Medical School, Madison, WI, USA, 2Illinois
Institute of Technology, Chicago, IL, USA.
Protein kinase A-mediated (PKA) phosphorylation of cardiac myosin binding
protein-C (cMyBP-C) accelerates the kinetics of cross-bridge cycling and ap-
pears to relieve the tether-like constraint of myosin heads imposed by
cMyBP-C (Colson et al., 2008, Circ Res., 103:244-251). We favor a mechanism
in which phosphorylation of the 3 PKA sites in cMyBP-C modulates cross-
bridge kinetics by regulating the proximity and interaction of myosin with
actin. To test this idea, we used synchrotron low-angle x-ray diffraction and
mechanical measurements in skinned myocardium isolated from a mouse
model with phosphomimetic substitutions in cMyBP-C, i.e., the CTSD mouse.
The substitutions were introduced by transgenic expression of cMyBP-C with
Ser-to-Asp mutations on a cMyBP-C null background. Western blots showed
that expression of CTSD cMyBP-C was 85% of wild-type (WT), and the heart
weight to body weight ratio was similar (5.2 5 0.2 mg/g) in CTSD and WT
mice. Expression of WT cMyBP-C on the knockout background served as con-
trol (i.e., the CTWT mouse). Skinned myocardium from CTSD and CTWT
mice exhibited similar maximum active forces (mN/mm

2
: 17.7 5 3.7 vs

13.2 5 2.9), Ca2þ-sensitivities of force (pCa50: 5.55 5 0.03 vs 5.58 5
0.04), and maximum rates of force development (ktr, sec

-1: 20.2 5 1.7 vs
22.5 5 1.9; kdf, sec

-1: 37.6 5 3.7 vs 43.2 5 2.3). I11/I10 intensity ratios and
d10 lattice spacings determined from equatorial reflections from CTSD and
CTWT myocardium were used to determine the effect of constitutive
cMyBP-C phosphorylation on the distribution of cross-bridge mass between
the thick and thin filaments and on interfilament lattice spacing. The results
suggest that interactions between cMyBP-C and the S2 domain of myosin
heavy chain are dynamically regulated by phosphorylations in the cMyBP-C
motif. (AHA-predoctoral fellowship (BAC); NIH-HL-R01-82900)

1196-Pos Board B40
Obscurin Interacts with a Novel Isoform of Myosin Binding Protein
C-Slow to Regulate the Assembly of Thick Filaments
Maegen A. Borzok, Rebecca Hu, Amber L. Bowman, John Strong,
Robert J. Bloch, Aikaterini Kontrogianni-Konstantopoulos.
University of Maryland, Baltimore, MD, USA.
Obscurin is a multidomain protein composed of adhesion and signaling do-
mains that plays key roles in the organization of contractile and membrane
structures in striated muscles. We used adenoviral-mediated gene transfer to
overexpress its extreme NH2-terminus in developing myofibers, followed by
immunofluorescence and ultrastructural methods to study its effects in sarco-
merogenesis. We found that overexpression of obscurin’s second immunoglob-
ulin domain (Ig2) inhibits the assembly of A- and M-bands, but not Z-disks and


	Decreased Fatigue Tolerance In Diaphragm Muscle Of Slow Troponin T Knockdown Mice
	Troponin Isoforms and Stretch-activation of Insect Flight Muscle
	Tracking of Qdot Conjugated Titin Antibodies in Single Myofibril Stretch Experiments Reveals Ig-domain Unfolding at Physiological Sarcomere Lengths
	Constitutive Phosphorylation of Cardiac Myosin Binding Protein-C Increases the Probability of Myosin Cross-bridge Interaction with Actin
	Obscurin Interacts with a Novel Isoform of Myosin Binding Protein C-Slow to Regulate the Assembly of Thick Filaments