Curtin, Nancy

Marston, Stteven B.

Song, Weihua

Woledge, Roger C.

Elsevier - Publisher Connector 

Monday, February 27, 2012 355atranslates to changes in contractility and calcium transients in isolated adult rat
cardiac myocytes. One day after cell isolation, we found an acute, dose-
dependent decrease in the peak height of contraction with tamoxifen concentra-
tions ranging from 1-10mM. This was accompanied by diminished calcium
transient amplitude. Additionally, the percentage of rod-shaped cells that visi-
bly contract dose-dependently decreased over the course of one hour of pacing
with tamoxifen. Raloxifene, also in the SERM class of drugs, had a pattern of
effects similar to tamoxifen. In conclusion, the acute tamoxifen and raloxifene-
induced inhibition of cardiac myocyte contractility may contribute to the tran-
sient cardiomyopathy seen in MCM transgenic mice. The results of this study
emphasize the importance of using the minimum dose of tamoxifen required for
gene excision in MCM transgenic mice, as well as incorporating appropriate
controls to address tamoxifen-mediated acute cardiomyopathy.
1800-Pos Board B570
Myofilament Dysfunction in the Infarct Border Zone
Rafael Shimkunas, Mona Bazargan, Om Makwana, Paul C. Simpson,
Mark B. Ratcliffe, Anthony J. Baker.
VA Medical Center & Univ. Calif. San Francisco, San Francisco, CA, USA.
Introduction: Soon after myocardial infarction, a poorly contracting border
zone forms adjacent to the infarct. Myocardium in the infarct border zone re-
mains normally perfused with blood, and the cause of border zone dysfunction
is unclear. Goal: Investigate the role of myofilament dysfunction in the im-
paired contraction of the infarct border zone. Methods: We studied sheep
hearts, 2 or 8 weeks after infarction of the apex of the heart and non-
operated controls. Myofilament contraction was assessed using in-vitro con-
tractions of skinned cardiac muscle fibers. Muscle fiber bundles were dissected
from the infarct border zone and from multiple regions ranging up to 6 cm from
the infarct. Skinned fiber force development was referenced to several mea-
sures of the content of contractile material within the samples. Results: In
the border zone immediately adjacent to the infarct, maximal force develop-
ment (Fmax) was reduced by 38 5 2% (n=7, P<0.001) compared with
Fmax of myocardium remote from the infarct. Fmax for remote zone myocar-
dium was similar to that for uninjured myocardium (~90 mN/mm2). The width
of the border zone was defined by a decreased Fmax that extended up to 5-6 cm
away from the infarct. Fmax rose in a linear gradient between the infarct and
the remote zone. There were no differences between border zone and remote
zone in: histological staining for collagen; the area fraction of myocardium oc-
cupied by myofilbrils (or mitochondria); or in the abundance of myosin. There-
fore, depressed border zone Fmax was not explained by decreased content of
contractile material. Moreover, treatment of skinned fibers with protein phos-
phatase 1 did not affect border zone Fmax, suggesting that myofilament protein
phosphorylation was not involved in border zone dysfunction. Conclusions:
Myofilament dysfunction contributes to impaired contraction in the infarct bor-
der zone.
1801-Pos Board B571
Dynamic Perturbations within the Ubiquitin Proteasome System in
Diabetic Cardiomyopathy Associated with Type 1 Diabetes Mellitus
Shannamar Dewey, Aldrin V. Gomes.
UC Davis, Davis, CA, USA.
Patients with type 1 diabetes mellitus (T1DM) typically have Diabetic Cardio-
myopathy (DCM) characterized by diastolic dysfunction and cardiac remodel-
ing. The ubiquitin proteasome system (UPS) is the primary proteolytic system
active in cardiac muscle protein degradation and has been shown to be activated
by hypoinsulinemia. The Akita mouse model offers a whole organism model of
T1DMwhich develops hypoinsulinemia and hyperglycemia in a timeline that is
analogous to the human disease. Cardiac tissue from mice that were pre-
diabetic (2 weeks old), recently diabetic (5 weeks old) and diabetic (12 weeks
old) was analyzed. At two weeks of age, with no hyperglycemia or cardiac at-
rophy, proteasome caspase-like, trypsin-like and chymotrypsin-like activities
were not altered. At five weeks of age, mice were hyperglycemic with de-
creased cardiac mass and had caspase-like and trypsin-like 26S activities that
were both suppressed approximately 15%. Interestingly, protein levels of the
RPT1 19S proteasome subunit were also decreased. At 12 weeks of age, the
20S caspase-like and trypsin-like activities were both increased by over 20%.
Lysosomal proteases showed a marked attenuation of activity at this time point.
Caspase-3 and calpain activity levels were not altered at any time. Immunoblot-
ting of the 20S subunit PSMA6 suggests an increase in the amount of 20S pro-
teasomes in diabetic hearts at this age. The inducible proteasome subunit,
MECL-1 also increased at this time point (P<0.06). RT-PCR showed that
the mRNA levels of the proteasome activator, PA28 alpha, and one 20S cata-
lytic subunit, PSMB5, were not changed at 12 weeks. These results suggest that
perturbations within cardiac proteolytic systems during T1DM are dynamic
with disease progression and the status of the UPS in this disease is complex,
likely involving multiple levels of regulation.1802-Pos Board B572
Reduced Efficiency of Intact Papillary Muscles in ACTC E99K Transgenic
Mouse Heart
Weihua Song, Stteven B. Marston, Roger C. Woledge, Nancy Curtin.
NHLI, Imperial College London, London, United Kingdom.
The E99K transgenic (TG) mouse expresses mutant cardiac actin as reported in
human patients being diagnosed with hypertrophic cardiomyopathy (HCM)
predominantly in the apex. The mouse has recapitulated many phenotypes as
in human patients including sudden cardiac death, apical hypertrophy, fibrosis,
myocyte disarray and higher Ca2þ-sensitivity. We are now reporting a reduced
efficiency of the mutant cardiac muscle comparing with their non-transgenic
(NTG) litter mates. Intact papillary muscle used in the experiments and the ef-
ficiency during contractions was measured with a protocol of 40 twitches in 20s
(27oC). The work and the heat production were measured during each set of 40
twitches and during runs with no stimulation as control. To mimic the cardiac
cycle, each stimulus was followed by an isometric period (isovolumic contrac-
tion) of 0.12s, shortening by 10%L0, an isometric period (isovolumic relaxa-
tion), and lengthening by 10% L0. Four movement velocities, 0.5, 0.67, 1
and 2 L0/s were tested. The isovoluminc relaxation periods were adjusted ac-
cordingly. Net work was calculated as the integral of active force and length
change. Heat production, an index of metabolic cost, was calculated from tem-
perature change measured with a thermopile of constantan-chromel thermocou-
ples. Muscle of TG mouse (n=12) did more work/mg muscle (33 5 1, mJ/g)
than muscles from NTG littermates (195 4, mJ/g, n=11), with disproportion-
ately more total energy, work þ heat (2635 10, mJ/g) than NTG (1145 18,
mJ/g). Thus E99K muscle was less efficient (= work/total energy), 0.127 5
0.005 than NTGmuscle, 0.1665 0.015. Considering the higher Ca2þ-sensitiv-
ity detected in myofibril and skinned papillary muscle levels, more energy was
likely used for active transport of ions such as Ca2þ.
1803-Pos Board B573
Post-Infarction Remodelling of Cardiac Muscle Assessed by Diffusion-
Weighted MR Imaging and Histological Methods
Mihaela Pop1, Nilesh Ghugre2, Beiping Qiang2, Qiang Xu3, Jen Barry2,
Lily Morikawa3, Elliot R. McVeigh4, Graham A. Wright1.
1University of Toronto, Toronto, ON, Canada, 2Sunnybrook Research
Institute, Toronto, ON, Canada, 3CMHD Pathology Core, Toronto, ON,
Canada, 4Johns Hopkins University, Dept. Biomedical Engineering,
Baltimore, MD, USA.
Cardiac magnetic resonance (MR) is a non-invasive imagingmethod that can be
used to assess important alterations in the structure and function of the ischemic
heart muscle. In this study, we used a pre-clinical swinemodel of chronic infarc-
tion generated by an occlusion-reperfusion method. The hearts from six animals
were explanted at ~5 weeks post-infarction, and imaged on a 1.5T GE SignaEx-
cite magnet using a high-resolution DW-MRI method (voxel size < 1mmt3).
In these images we delineated necrotic core scar (CS) areas and peri-infarct (PI)
areas, where fractional anisotropy and apparent diffusion coefficient had signif-
icantly altered values compared to those in remote, healthy myocardium. Rep-
resentative samples cut in short-axis, underwent histo-pathological analysis. In
order to evaluate the severity of structural changes due to fibrosis, quantitative
histological analysis using Sirius Red stain was performed. This demonstrated
very good correspondence between the areas scored as dense collagen
(>75%) as well as intermediate collagen density (25-75%, where fibrotic zones
intermingled with viable myocytes), with the CS areas and PI areas, respec-
tively, identified in MR images. Furthermore, to evaluate the changes in electri-
cal function in the heterogeneously remodelled areas, we employed light
micrographs of connexin Cx43 (which is responsible for cell-to-cell electrical
coupling) prepared using imunohistochemistry methods, as well as fluorescence
micrographs of Cx43. The analysis of fluorescence images demonstrated a dis-
turbed pattern of gap junctions and a reduction of Cx43 density in the PI areas
compared to areas selected from healthy myocardial tissue. The structural and
functional remodelling of the cardiac muscle in the post-infarction period often
underlie malignant arrhythmic events; therefore, the localization (particularly
deep in the myocardium) and characterization of the CS and PI areas by means
of a non-distructive way (such as DW-MR imaging) is extremely valuable.
1804-Pos Board B574
A Model of Hypertrophic Cardiomyopathy Induced by a Protein Tyrosine
Phosphatase SHP2 Mutation Demonstrates Increased Myofibrillar
Function
Kerry S. McDonald, Laurin M. Hanft, Maike Krenz.
University of Missouri, Columbia, MO, USA.
In the majority of cases, hypertrophic cardiomyopathy (HCM) is a genetic dis-
ease of the sarcomere or sarcomere-related proteins. Many of these mutations
have been shown to directly affect sarcomere contractile performance. How-
ever, mutations in other, non-sarcomeric proteins can also cause HCM. For


Reduced Efficiency of Intact Papillary Muscles in ACTC E99K Transgenic Mouse Heart 

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Reduced Efficiency of Intact Papillary Muscles in ACTC E99K Transgenic Mouse Heart

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