Ackermann, Maegen A.

Contreras, Minerva

Hecker, Peter A.

Hu, Li-Yen R.

Kontrogianni-Konstantopoulos, Aikaterini

O'Connell, Kelly A.

Perry, Nicole A.

Shriver, Marey

Stanley, William C.

Elsevier - Publisher Connector 

Monday, February 4, 2013 187a960-Plat
Structural Dynamics of Actin-Myosin Bound and Unbound States of
Cardiac Myosin Binding Protein-C Detected by Dipolar EPR
Brett A. Colson, Zachary M. James, David D. Thomas.
University of Minnesota, Minneapolis, MN, USA.
We have used site-directed spin labeling and pulsed dipolar electron-electron
paramagnetic resonance (DEER) to resolve the structure and dynamics of flex-
ible and disordered regions of myosin binding protein-C (MyBP-C)’s cardiac
isoform, with implications for the pathophysiology of hypertrophic cardiomy-
opathy (HCM). N-terminal domains of cMyBP-C contain binding domains for
several interaction partners in the myofilament, including myosin heavy chain
subfragment 2 (S2) and actin. We engineered pairs of labeling sites in protein
fragments of mouse cMyBP-C to measure with high resolution distance and
disorder between (1) domains C0 and C1, flanking the flexible Pro/Ala-rich
linker, and between (2) domains C1 and C2, flanking the partially disordered
phosphorylation motif, using DEER. Changes in distance and disorder were as-
sessed for double-Cys mutant cMyBP-C’s free in solution and when bound to
myosin S2 or actin, with or without cMyBP-C phosphorylation by protein ki-
nase A (PKA). Understanding conformational transitions in the flexible and dy-
namic portions of cMyBP-C upon actin-myosin binding and phosphorylation
provide new molecular insight into defining its modulatory role in muscle force
development. (NIH-F32 to BAC; NIH-R01 to DDT)
961-Plat
Obscurin: A New Player in Cardiac Hypertrophy
Maegen A. Ackermann, Li-Yen R. Hu, Peter A. Hecker, Minerva Contreras,
Nicole A. Perry, Marey Shriver, Kelly A. O’Connell, William C. Stanley,
Aikaterini Kontrogianni-Konstantopoulos.
University of Maryland, Baltimore, MD, USA.
Obscurins, encoded by the single OBSCN gene, comprise a family of giant
(~890-810 kDa) and small (~550-50 kDa) proteins of vertebrate striated mus-
cles composed of adhesion and signaling motifs. Giant obscurins intimately
surround sarcomeres at the level of M-bands and Z-disks where they participate
in the assembly, stabilization, and integration of the contractile cytoskeleton
with other sarcoplasmic structures. Consistent with this, the immunoglobulin
(Ig) domains 58 and 59 of obscurins interact directly with Ig domains 9 and
10 of titin located at the periphery of Z-disks. Genomic linkage analysis has re-
cently revealed a missense mutation (R4344Q) in obscurin Ig58 that is causally
linked to hypertrophic cardiomyopathy (HCM). To examine how the R4344Q
mutation leads to the development of HCM, we generated two animal models:
a knock-in model that contains full length obscurins carrying the R4344Q mu-
tation, and a partial knock-out model that lacks Ig 58 and 59. Immunoblot and
immunofluorescence analysis indicated that both mutant and truncated obscur-
ins are readily expressed in cardiac muscles, and properly incorporated into sar-
comeres. Homozygous partial knock-out mice developed overt cardiac
hypertrophy by 12 months of age, as measured by echocardiography; notably,
hypertrophy was exacerbated in the female homozygous animals. While a hy-
pertrophic trend was apparent in homozygous knock-in animals, phenotypic
and functional alterations of the affected hearts were not statistically significant
from those of wild-type animals. Importantly, trans-aortic constriction of
~2 months old knock-in and partial knock-out male and female mice led to se-
vere cardiac hypertrophy within 4-8 weeks post-surgery, as evaluated by echo-
cardiography. We are currently examining the cellular and biochemical
manifestations of mutant and truncated obscurins, as related to the development
of HCM. Our studies provide the first in vivo models to study the molecular de-
fects that underlie HCM due to altered obscurins.
962-Plat
The HCM-Associated Cardiac Troponin T Mutation K280N Increases the
Energetic Cost of Tension Generation in Human Cardiac Myofibrils
Claudia Ferrara1, E. Rosalie Witjas-Paalberends2, Nicoletta Piroddi1,
Beatrice Scellini1, Chiara Tesi1, Vasco Sequiera2, Cristobal dos Remedios3,
Saskia Schlossarek4, Judy Leung5, Lucie Carrier4, Charles Redwood6,
Steve Marston5, Jolanda van der Velden2, Corrado Poggesi1.
1Universita` di Firenze, Firenze, Italy, 2VUMC, Amsterdam, Netherlands,
3University of Sidney, Sidney, Australia, 4University Medical Center
Hamburg-Eppendorf, Hamburg, Germany, 5Imperial College, London,
United Kingdom, 6University of Oxford, Oxford, United Kingdom.
A novel homozygous mutation in the TNNT2 gene encoding cardiac troponin T
(cTnT K280N) was identified in one HCM patient undergoing cardiac trans-
plantation. mRNA and Mass Spectrometry analyses revealed expression of
the mutant alleles without evidence of haploinsufficiency. Kinetics of contrac-
tion and relaxation of myofibrils from a frozen left ventricular sample of the
K280NHCM patient were compared to those of ‘‘control’’ myofibrils (from do-
nor hearts, from aortic stenosis patients, and from HCM patients negative forsarcomeric protein mutations). Preparations, mounted in a force recording ap-
paratus (15 C), were maximally Ca2þ-activated (pCa 4.5) and fully relaxed
(pCa 9) by rapid (<10 ms) solution switching. The rate constant of active ten-
sion generation following maximal Ca2þ activation (kACT) was markedly faster
in K280N myofibrils (ca. 1.7 s1) compared to controls (0.7-1 s1). The rate
constant of isometric relaxation (slow kREL) was 2-3-fold faster in K280Nmyo-
fibrils than in controls, evidence that the apparent rate with which cross-bridges
leave the force generating states is accelerated in the mutant preparations. The
results suggest that the energy cost of tension generation is increased in the
K280N sarcomeres. Simultaneous measurements of maximal isometric
ATPase and Ca2þ-activated force in Triton-permeabilized left ventricular mus-
cle strips from the K280N sample demonstrated that tension cost was much
higher in the K280N than in control myocardium. Replacement of the mutant
protein by exchange with wild-type recombinant human Tn in the K280Nmyo-
fibrils reduced both kACT and slow kREL close to control values. This indicates
that the HCM-associated TNNT2 mutation K280N primarily alters apparent
cross-bridge kinetics and impairs sarcomere energetics. Supported by the 7th
Framework Program of the European Union, ‘‘BIG-HEART’’ grant agreement
241577.
963-Plat
Additive Compensatory Effects of Cardiac Troponin I and Cardiac Tro-
ponin T N-Terminal Truncations on the Disease Phenotypes of a Familial
Hypertrophic Cardiomyopathy Mutation (E180G) of a-Tropomyosin
Hanzhong Feng1, David F. Wieczorek2, Jian-Ping Jin1.
1Department of Physiology, Wayne State University School of Medicine,
Detroit, MI, USA, 2Department of Molecular Genetics, Biochemistry and
Microbiology, University of Cincinnati, Cincinnati, OH, USA.
A naturally occurring proteolytic N-terminal truncation of cardiac troponin I
(cTnI-ND) has been shown to compensate for functional abnormalities caused
by cardiomyopathic mutations. A proteolytic N-terminal truncation of cardiac
TnT (cTnT-ND) elongates ventricular ejection time to compensate for cardiac
output against high pressure load. To investigate the mechanisms for these tro-
ponin modifications to modulate the function of cardiac muscle thin filament,
we produced transgenic mouse lines co-expressing cTnI-ND and/or cTnT-
ND with a family hypertrophic cardiomyopathy mutation of cardiac a-tropo-
myosin (TM-E180G). The over expression of TM-E180G resulted in ~90%
replacement of endogenous a-tropomyosin in the cardiac muscle with normal
total stoichiometry. Functional studies in ex vivo working hearts of 2-month-
old TM-E180G mice showed lower diastolic velocity and higher left ventricu-
lar diastolic pressure, indicating higher Ca2þ sensitivity. b-MHC expression
together with myocardial fibrosis was found in the left ventricle of 28-day
and 2-month-old TM-E180G mice with early failing phenotypes (lower stroke
volume and lower systolic velocity). In double and triple transgenic mouse
hearts, expression of cTnI-ND and/or cTnT-ND decreased the occurrence of
b-MHC in TM-E180G mouse hearts. Functional studies showed cTnI-
NDþcTnT-ND did not override the contractile phenotype of TM E180G
mutation but had beneficial effects on improving stroke volume and reducing
fibrosis, with additive effects in the triple transgenic hearts. While TM-
E180G mice usually die between 4 and 5 months of age, cardiac function of
TM-E180GþcTnI-NDþcTnT-ND triple transgenic mice remained apparently
normal at 10-11 months of age as shown by the compensated heart function
and cardiac efficiency with minimal b-MHC occurrence. These results demon-
strated compensatory effects of posttranslational modifications of troponin on
the functional abnormality of tropomyosin for potential applications in the
treatment of heart failure.
964-Plat
Effects of Cardiomyopathy-Related Troponin T Mutations on the
Ubiquitin-Proteasome System
Jennifer E. Gilda, Ziyou Cui, Shannamar Dewey, Aldrin V. Gomes.
University of California, Davis, Davis, CA, USA.
Familial hypertrophic cardiomyopathy (FHC) is a disease of the myocardium
that can be caused by a mutation in a sarcomeric gene. TNNT2 encodes cardiac
troponin T (cTnT), a sarcomeric protein important for cardiac muscle contrac-
tion. Mutations in TNNT2 are frequently linked to sudden cardiac death (SCD);
however their role in heart failure and cardiomyopathy is unclear. We hypoth-
esize that changes in the ubiquitin-proteasome system (UPS) are related to in-
creased risk of cardiac death, and that changes in the UPS may be related to
calcium sensitization caused by TNNT2 mutations. We examined two muta-
tions in TNNT2, R278C, which is associated with mild effects and late-onset
heart disease, and the calcium-sensitizing mutation I79N, which is early-
onset and has severe cardiac effects, including SCD. In 3-month-old transgenic
mice expressing the I79N mutant form of troponin T, expression of the protea-
some subunit PSMA6was decreased by approximately 35% in I79Nmice when


Obscurin: A New Player in Cardiac Hypertrophy 

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Obscurin: A New Player in Cardiac Hypertrophy

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