fold, respectively) and unchanged in FF-treated hearts.

Perfused working hearts from FF-treated mice showed

decrease FA oxidation by 40%, a finding that could be

explained by reduced plasma FA supply. In hearts from TTA-

treated mice, however, FA oxidation was maintained despite a

reduced FA supply. We propose therefore that TTA may have

a direct effect on the heart that compensates for the indirect

effect of a reduced FA supply.

doi:10.1016/j.yjmcc.2006.03.046

032. Changing myocardial energy utilization from fat to

glucose improves post-ischemic recovery in hearts from

type 2 diabetic (db/db) mice

Anne D. Hafstad, Ahmed M. Khalid, Ole-Jakob How, Terje S.

Larsen, Ellen Aasum. Institute of Medical Biology, Faculty of

Medicine, University of Tromsø, Norway

Hearts from type 2 diabetic db/db mice demonstrate over-

reliance on fatty acid oxidation (FAox) for energy production, as

well as reduced functional recovery following ischemia–

reperfusion. Although db/db mice show an overall insulin

resistance in vivo, we recently reported that insulin induces a

marked shift towards glucose oxidation (GLUox) in perfused db/

db hearts. We therefore hypothesized that such a shift in

metabolism should improve post-ischemic recovery in this

model. Hearts from non-diabetic (db/+) and diabetic mice were

perfused with 0.7 mM palmitate and either 5 mM glucose and no

insulin (LG), 5 mM glucose + 0.3 mU/ml insulin (LGI) or 33

mM glucose + 0.9 mU/ml insulin (HGHI). In addition to pre-

ischemic myocardial FAox and GLUox, ventricular function

was measured before and after 40 min low-flow ischemia. While

insulin and high glucose had only modest effects on metabolism

and recovery in db/+ hearts, it produced a marked metabolic

shift towards GLUox in db/db hearts, which was associated with

a significant improvement in functional recovery (Table). These

results suggest that a metabolic shift in favor of glucose could

explain the beneficial effect of glucose-insulin-potassium

therapy in diabetes.

doi:10.1016/j.yjmcc.2006.03.047

033. The impact of increasing calcium on myocardial

function in experimental uraemia

Dunja Aksentijevic a, Sunil Bhandari b, Anne-Marie L.

Seymour a. a Department of Biological Sciences, University of

Hull, Hull, UK. b Department of Renal Medicine, Hull Royal

Infirmary, Hull, UK

Cardiovascular complications are the leading cause of

mortality in chronic kidney disease (CKD), accounting for

50% of all deaths among patients with end-stage renal failure.

However, the mechanisms underlying this cardiac dysfunction

have yet to be elucidated. The aim of this study was to evaluate

the effect of increasing extracellular calcium on cardiac

physiology and function in experimental uraemia. Uraemia

was surgically induced in male Sprague–Dawley rats via a two

stage sub-total nephrectomy. Six weeks post-surgery, following

equilibration with 1.25 mM [Ca2+]i, isolated hearts were

perfused with high calcium (2.5 mM) K-H buffer in the

isovolumic mode, with simultaneous monitoring of cardiac

function and myocardial oxygen consumption. At 6 weeks, sub-

totally nephrectomized animals were significantly uraemic (urea

14 T 3 mmol/L vs. 6 T 1 mmol/L P < 0.05 n = 5; creatinine 64 T8 vs. 40 T 11 Amol/l P < 0.05 n = 5), hypertensive (systolic

pressure 159 T 22 vs. 151 T 21 mm Hg P < 0.01 n = 25) and

hyperinsulinaemic (38% increase in circulating insulin levels).

Hearts from uraemic animals display a blunted functional

response to increased [Ca2+] compared to controls, with an

11% decrease in contractile function (RPP 37 T 11� 103 vs. 41 T17 � 103 mm Hg min�1), and 3% increase in oxygen

consumption (2.8 T 0.5 vs. 2.7 T 0.3 Amol O2/min/g tissue)

and a markedly reduced cardiac efficiency (n = 5). However, at

this stage, there was no evidence of LV hypertrophy. Therefore,

at 6 weeks, uraemia is characterized by significant hemody-

namic abnormalities, hyperinsulinemia and indication of altered

cardiac calcium handling and contractile function.

doi:10.1016/j.yjmcc.2006.03.048

034. Impact of chronic gliclazide therapy in the nuclear

oxidative stress and insulin-resistance levels in an animal

model of diabetes

Pedro Monteiro a, Elsa Nunes b, Raquel Seica b,

Lino Goncalves a, Luıs A. Providencia a. a Basic Research Unit

In Cardiology-Cardiology Department, Portugal. b Physiology

Department, Coimbra Medical School, Coimbra, Portugal

Introduction: Type 2 diabetes (DM) is associated with a

higher incidence and worse prognosis of ischemic heart

disease. High levels of insulin-resistance and nuclear oxidative

stress, common in DM, may be involved. However, the

mechanisms of these changes in DM and their modulation by

chronic administration of gliclazide (a sulphonylurea) are not

yet fully known.

Aim: To evaluate, in an animal model of DM, if chronic

administration of gliclazide changes insulin-resistance and

nuclear oxidative stress levels.

Cardiac

metabolism

(Amol/min/g

dry wt)

Cardiac output

GLUox FAox Pre-ischemic

(ml/min)

Post-ischemic

rec. (%)

db/+ LG 2.0 T 0.1 0.8 T 0.2 11.8 T 0.7 65.2 T 5.4

LGI 2.3 T 0.1 0.7 T 0.2 12.1 T 05 69.7 T 5.8

HGHI 2.5 T 0.2* 0.4 T 0.1* 12.4 T 0.9 81.1 T 5.7

db/db LG 0.5 T 0.1# 2.3 T 0.2# 8.2 T 0.6# 32.6 T 5.8#

LGI 1.1 T 0.1*# 1.3 T 0.1*# 9.8 T 0.4# 51.1 T 5.4*#

HGHI 1.9 T 0.2*# 0.4 T 0.1* 8.8 T 0.6# 73.4 T 6.6*

* P < 0.05 vs. LG.# P < 0.05 vs. db/+.

ABSTRACTS / Journal of Molecular and Cellular Cardiology 40 (2006) 920–1015932

Material and methods: Two experimental groups (n = 12/

group)-GK, with Goto-Kakizaki rats (animal model of DM)

and GK S, (GK rats treated with gliclazide 10 mg/kg/day

between 12 and 16 weeks of age). At 12 and 16 weeks,

insulin plasma levels were determined, using competitive

enzyme-linked imunosorbent assay (ELISA) and used to

calculate logarithm of the homeostasis model assessment

[log(HOMA)], a measure of insulin-resistance, according to

the formula log[(Glicemia (mmol/l) � Insulinemia (AU/ml))/

22.5].

At 16 weeks, 24 h urine was collected to determine levels of

8-hidroxy-2V-desoxyguanosine (8-OHdG), a measure of DNA

oxidative stress, using competitive ELISA.

Results: Log(HOMA) was significantly decreased in the GK

S group between 12 and 16 weeks (0.07 T 0.01 versus 0.08 T0.01; P < 0.05), so that at 16 weeks it was significantly lower

than that of the GK group (0.08 T 0.01 versus 0.09 T 0.02; P <

0.05). Regarding 8-OHdG urine levels, there was a trend

towards a lower value in the GK S group (155 T 42 versus

183 T 69 ng/24 h; P < 0.05)—Fig. 1.

Conclusion: In diabetic animals, chronic therapy with

gliclazide decreases of insulin-resistance and tends to lower

nuclear oxidative stress level.

doi:10.1016/j.yjmcc.2006.03.049

035. Oxidative capacity recovers in the unloaded heart with

clenbuterol

Kameljit K. Kalsi, Gopal K.R. Soppa, Cesare M.N. Terracciano,

Ryszard T. Smolenski, Magdi H. Yacoub. Imperial College

London, Heart Science Centre, Harefield, Middlesex, UK

Long-term mechanical unloading of the heart by LV assist

device (LVAD) causes left ventricular (LV) atrophy in patients

with heart failure. It has been proposed that LV atrophy may be

prevented by administration of the h2-agonist clenbuterol.

Chronic administration of clenbuterol induces myocardial

hypertrophy and improves calcium handling. However, its

actions on the metabolism of the unloaded myocardium are

unclear. We hypothesize that clenbuterol induces functional

improvement by increasing oxidative metabolism. Hearts from

Lewis rats were mechanically unloaded using heterotopic

abdominal transplantation. Osmotic mini-pumps, inserted at

the time of transplantation, administered either saline, control

group (n = 4) or clenbuterol (CLEN) (2 mg/kg) (n = 5). After 1

week, the transplanted hearts were excised and cardiomyocytes

isolated. Cells were incubated in Krebs buffer containing 11

mM 1–13C glucose for 2 h. Glucose oxidation was determined

by using liquid chromatography/mass spectrometry glutamate

to alanine isotopomer ratios. Non-oxidative metabolism of

glucose was measured by the release of 13C-enriched lactate in

the media. Glutamate/alanine in control cardiomyocytes was

51 T 2% but decreased in unloaded cells 41 T 4% (P < 0.05).

Glucose oxidation increased with CLEN in both recipient and

unloaded cells to (62 T 3% and 64 T 9% respectively). Lactate

enrichment increased in control unloaded cardiomyocytes to

62 T 6% compared to recipient cells 48 T 1%. However, with

CLEN lactate, levels were similar in recipient and unloaded

cardiomyocytes (59 T 1% and 57 T 1%, respectively). In

conclusion, clenbuterol improves glucose oxidation in the

unloaded myocardium and this may be useful during LVAD

treatment in patients with heart failure.

doi:10.1016/j.yjmcc.2006.03.050

036. Diazoxide increases cytosolic ATP: A new paradigm

for preconditioning?

Sophie Pelloux a, Brigitte Chhin a, Carlos Ojeda b,

Be Wieringa c, Michel Ovize a, Yves Tourneur a. a Inserm

E0226, France. b Inserm Erit-M 107, France. c NCMLS, The

Netherlands

We developed a cardiac cell line model derived from the

HL-1 to study the induction of pharmacological precondition-

ing by diazoxide (100 AM) against simulated ischemia–

reperfusion. We reported earlier that HL-1 cells possess

electrophysiological properties of cardiac myocytes, express

cardiac specific proteins, and that diazoxide was able to

attenuate HL-1 cell death following a prolonged hypoxia and

reoxygenation.

In the present study, cells were transfected to transiently

express the firefly luciferase with a modified pCDNA3

plasmid. In the presence of luciferin (200 AM), cytoplasmic

ATP could be monitored by photon counting. Diazoxide did

not change the global ATP concentration measured by

biochemical techniques (2.65 + �0.12 nmol/mg prot. vs.

control 2.71 + �0.07, N = 8), which indicates that ATP is not

homogeneous and that luciferase is sensitive to local concen-

tration modifications. Luminescence rapidly responded to

changes in oxidative phosphorylation or glycolysis. Applica-

tion of diazoxide (100 AM) produced a rapid and prolonged

increase in ATP. This increased ATP production was recorded

even in the presence of azide (2 mM), a blocker of the electron

transport chain, and was prevented by glybenclamide (10 AM).

These data suggest that diazoxide may protect HL-1

cardiomyocytes rather by activating glycolysis than via a

direct effect on mitochondria.

doi:10.1016/j.yjmcc.2006.03.051

037. Cardiac performance is reduced following high-fat diet

Ashwin Akki, Anne-Marie L. Seymour. Department Biological

Sciences, University of Hull, UK

Cardiac hypertrophy results in cellular remodelling of the

heart and, in particular, in alterations in metabolism, which

may underlie the development of cardiac dysfunction. One

key characteristic of this remodelling is a marked reduction

in fatty acid oxidation. We have investigated whether a

mismatch between fatty acid supply and utilization occurs in

cardiac hypertrophy that may predispose the heart to an

ABSTRACTS / Journal of Molecular and Cellular Cardiology 40 (2006) 920–1015 933