junction channel protein, connexin-43, which need further

analysis.

doi:10.1016/j.yjmcc.2006.03.065

051. Nuclear transcription factors, NFKB and NFAT c4

were activated by autoantibodies (AAB) in neonatal rat

heart cells under different culture conditions

Okruhlicova Ludmila a, Schulze Wolfgang b, Bartel Sabine b,

Wallukat Gerd b. a Institute for Heart Research SAS,

Bratislava, Slovakia. b Max-Delbrueck-Center, Berlin, Germany

Nuclear factors NFnB and calcineurine-NFATc4 are tran-

scription factors that have been implicated as critical intracel-

lular regulators of the cardiac hypertrophic growth response. In

resting cells, both factors remain in cytoplasm. Upon stimula-

tion, they translocate into nucleus and bind to DNA-binding

sites in the promoter region of genes.

In sera of patients with essential hypertension, pre-

eclampsia and various forms of dilated cardiomyopathy

autoantibodies (AAB) against G-protein-coupled receptors

were found. These AAB are able to disturb physiological

processes of the myocardium leading to its functional and

structural modifications. The aim of this study was to

examine the effect of patients’ AAB against alpha1-

adrenoceptor and AT1-receptor on nuclear translocation of

both factors in cultured neonatal rat cardiomyocytes.

Immunofluorescence showed that NFATc4 in contrast to

NFnB was localized in the nucleus of myocytes cultured in

AAB-free complete SM20 medium (1.2 mmol/l Ca2+).

Cytoplasmic localization of NFATc4 was observed in

myocytes cultured in Ca-free medium for 1 h. Treatment

of cells with AAB (1:40) for 1 h in complete medium

resulted in NFnB translocation into the nucleus. In contrast

AAB-induced translocation of NFATc4 was observed in

cells growing in Ca-free medium only. Our results indicate

calcium-dependent differences in NFATc4 and NFnB local-

ization in cultured neonatal rat myocytes and the ability of

patients’ AAB to induce translocation of both transcription

factors.

doi:10.1016/j.yjmcc.2006.03.066

052. Evidence for reduced troponin I phosphorylation and

altered troponin function in patients with hypertrophic

obstructive cardiomyopathy

Adam Jacques a, Andrew Messer a, Victor Tsang b, William

McKenna b, Steven Marston a. a Imperial College London,

London, UK. b The Heart Hospital, London, UK

Troponin was isolated from 5 non-failing donor hearts

and from 5 patients with HCM after septal myectomy (mean

septal wall thickness = 23.1 T 2.7 mm; mean left ventricular

outflow tract gradient = 105.2 T 18.1 mmHg). Phosphory-

lation levels of human cardiac TnI and troponin T (TnT)

were assayed in SDS-PAGE gels using Pro-Q diamond

phosphoprotein stain normalised to total protein in each

band. TnT from non-failing heart contained 3.05 T 0.2

molPi/mol and TnI contained 2.25 T 0.36 molPi/mol. In

HCM, the level of TnT phosphorylation was the same (3.11 T0.42 molPi/mol) but the level of TnI phosphorylation was

very low (0.11 T 0.15 molPi/mol, n = 5, P < 0.01) with 3

of the 5 HCM TnI samples showing no detectable

phosphorylation. Troponin samples were tested in thin

filaments reconstituted with human cardiac tropomyosin

and rabbit skeletal actin by in vitro motility assay.

Comparison of thin filaments from non-failing and HCM

heart troponin in dual chamber motility cells demonstrated

no difference in filament sliding speed at maximally

activating Ca2+ concentrations and no change in Ca2+

sensitivity despite the highly significant difference in TnI

phosphorylation. In view of this unexpected observation,

non-failing and HCM troponins were dephosphorylated with

acid phosphatase. This produced a marked increase in Ca2+

sensitivity and a 15% decrease in filament sliding speed in

non-failing compared to HOCM troponin. Therefore, HCM

muscle samples show reduced troponin I phosphorylation

and a new molecular phenotype for unphosphorylated

troponin. This is unlikely to be a direct effect of HCM-

causing mutations but could be a secondary effect of a

mutation or a direct response to pressure overload.

doi:10.1016/j.yjmcc.2006.03.067

053. Phosphorylation state of phospholemman at serine 68

regulates Na/K ATPase activity

Davor Pavlovic, Linda M. McLatchie, Michael J. Shattock,

William Fuller. Cardiovascular Division, The Rayne Institute,

King’s College London, UK

Phospholemman (PLM) is a small transmembrane protein

predominantly expressed in the heart, skeletal muscle and

brain. It is a member of the FXYD family of proteins that

associate with and regulate the Na/K ATPase (NKA).

Protein kinase A (PKA) phosphorylates PLM at serine 68

(S68); however, the effects of phosphorylation on NKA

activity are not fully characterised. The objectives of this

study were to characterise NKA currents in PLM wild type

(WT) and knockout (KO) mouse ventricular myocytes,

investigate the effects of a PLM peptide on these currents

and determine the effect of PKA phosphorylation of this

peptide.

NKA pump currents were measured using the whole-cell

patch-clamp technique. Peptides representing the 19 C-terminal

residues of PLM and its scrambled analogue were synthesised

and introduced into the myocyte interior through the patch

pipette. K-sensitive NKA currents were higher in KO myocytes

(2.87 T 0.12 pA/pF; n = 4) compared to WT (1.96 T 0.34 pA/pF;n = 4). 4 AM of unphosphorylated PLM peptide reduced the

currents inWT (from 2.15 T 0.11 to 1.42 T 0.23 pA/pF; P < 0.05,

n = 7) and KO (from 2.94 T 0.09 to 1.72 T 0.21 pA/pF; P < 0.05,

ABSTRACTS / Journal of Molecular and Cellular Cardiology 40 (2006) 920–1015 939

n = 5) myocytes. In contrast, 1 AM of S68 phosphorylated PLM

peptide increased current in WT (from 1.89 T 0.07 to 3.17 T 0.46pA/pF; P < 0.05, n = 6) and KO (from 2.83 T 0.12 to 3.81 = 0.26

fspA/pF; P < 0.05, n = 6) myocytes. No detectable changes in

NKA currents were observed in the presence of 4 AM of the

scrambled peptide. Coimmunoprecipitation studies demonstrat-

ed that both S68 phosphorylated and unphosphorylated PLM

peptides associate with NKA a1 but not with the structurally

related protein SERCA2a.

We conclude that unphosphorylated PLM inhibits NKA,

whereas serine 68 phosphorylated PLM stimulates NKA.

doi:10.1016/j.yjmcc.2006.03.068

054. Changes in thyroid hormone signaling contributes to

fetal programming after myocardial infarction in rats

I. Mourouzis, K. Markakis, T.h. Saranteas, A. Dimopoulos, S.

Tzeis, M. Panagiotou, C. Pantos, D.V. Cokkinos.

1st Cardiology Department, Onassis Cardiac Surgery Center,

Department of Pharmacology, University of Athens, Greece

Introduction: Fetal programming, in which a hypothyroid

phenotype is created, is recapitulated after myocardial

infarction (MI) with potential physiological consequences.

In this study, we investigated whether cardiac remodeling

after MI involves changes in thyroid hormone signaling.

Methods: Wistar rats were subjected to left coronary artery

ligation (AMI, n = 7), or sham operation (SHAM, n = 7).

Furthermore, hypothyroidism was induced in rats by admin-

istration of propylthiouracil for 21d (HYPO, n = 8) while non-

treated rats served as controls (CON, n = 8). After 8 weeks,

hearts were perfused in Langendorff mode for resting

contractile function measurements: left ventricular developed

pressure (LVDP in mm Hg) and +dp/dt and �dp/dt (in mm

Hg/s). T3 and T4 were measured in plasma. Furthermore,

thyroid hormone receptors a1 and h1 (TRa1, TRh1) in non-

infarcted myocardium and thyroid hormone responsive genes

such as Na+/H+ exchanger (NHE), SR Ca2+-ATPase (SERCA)

and PKC( were measured by Western blotting.

Results: Basal contractile function was significantly reduced

in both AMI and HYPO groups as shown in the table.

T3 and T4 plasma levels were not significantly different in

AMI and SHAM hearts. T3 and T4were 0.87 (0.04) and 52.5

(2.6) in CON vs. 0.23 (0.05) and 19.9 (0.38) in HYPO rats,

respectively, P < 0.05. TRa1 and TRh1 were found to be

downregulated 1.3 and 1.8-fold less in AMI than in SHAM

hearts, P < 0.05. SERCA and NHE1 expression was 2.1-

and 1.8-fold less in AMI than in SHAM, P < 0.05. PKC(was 1.3 more in AMI compared to SHAM, P < 0.05. In

HYPO hearts, SERCA was 2.3-fold less as compared to

CON hearts, P < 0.05. NHE1 was expressed 2.0-fold less

and PKC( 1.45-fold more in HYPO as compared to CON hearts,

P < 0.05.

Conclusion: Cardiac remodeling after MI results in tissue

hypothyroidism and this may be of important clinical and

therapeutic relevance.

doi:10.1016/j.yjmcc.2006.03.069

055. The tetrapeptide Ac-SDKP prevents interstitial

cardiac fibrosis in cardiomyopathic hamsters

H. Mongue-Din a, J.-M. Liu b, A. Salmon a, M.Y. Fiszman a,

J. Wdzieczak-Bakala b, Y. Fromes a. a Institut de

Myologie-INSERM U582, Paris. b ICSN, CNRS,

Gif-sur-Yvette, Paris

CHF147 hamster strain, a model for a myopathy with

cardiac involvement, develops a cardiopathy characterised

by focal loss of cardiocytes replaced by focal fibrosis.

Furthermore, this cardiopathy evolves towards a dilated

phenotype ending up in heart failure. Moreover, in failing

hearts, the development of interstitial fibrosis is responsible

for electrical activity impairments and loss of myocardial

compliance. A substrate for angiotensin-converting enzyme,

the hemoregulatory tetrapeptide Ac-SDKP, may present

antifibrotic properties. Normally present in blood at nano-

molar concentrations, the aim of this study was to

investigate the effects of pharmacological doses of Ac-

SDKP on structural modifications of the myocardium of

young cardiomyopathic hamsters.

Four groups of 4 months old CHF147 hamsters were

treated during 42 days with 3 different doses of Ac-

SDKP or placebo, using osmotic minipumps implanted

intraperitoneally. Animals were then sacrificed and hearts

were harvested for morphological analysis. Weight and

size of the ventricles were comparable in all groups.

Ventricular fibrosis was quantified on Sirius red stained

cryostat sections. Whereas fibrotic scar tissue remained

comparable, interstitial fibrosis was significantly less

pronounced in treated groups. This difference appeared

to be more important for the lowest dose of Ac-SDKP.

Histological results were confirmed by immunohistoche-

mistery. Furthermore TGF-h expression in ventricles was

analysed.

Thus, in a cardiomyopathic hamster model, a pro-

longed infusion of low dose Ac-SDKP demonstrated a

decreased progression of interstitial ventricular fibrosis.

Therefore, Ac-SDKP might be of therapeutic interest in

heart failure.

doi:10.1016/j.yjmcc.2006.03.070

LVDP +dp/dt �dp/dtAMI 83.6 (7.9) 2636 (354) 1587 (145)

SHAM 125.4 (4.9)* 3798 (174)* 2275 (66)*

HYPO 103.8 (2.3) 3200 (259) 1731 (85)

CON 129.3 (4.5)** 4476 (227)** 2461 (149)**

* P < 0.05 vs. AMI.

** P < 0.05 vs. HYPO.

ABSTRACTS / Journal of Molecular and Cellular Cardiology 40 (2006) 920–1015940