PhD School of Heart Research
4th Annual CHFR Symposium
PROGRAM
Thursday October 12, 2006
08.00 - 16.50
Holmenkollen Park Hotel Rica
Oslo, Norway
2
Welcome to the 4th Annual CHFR Symposium
Center for Heart Failure Research (CHFR) constitutes a branch of
medical research at the Faculty of Medicine at the University of Oslo,
Norway. It is also supported by funding from the Eastern Norway
Regional Health Authority (Helse Øst). The center represents a
concerted action of groups with complementary expertise that combines
state-of-the-art gene technology and capabilities to investigate protein
function in experimental systems. The systems range from isolated cells
and multicellular preparations to integrative physiology in
pathophysiological models of heart failure. The clinical research groups
of the Center for Heart Failure Research allow for testing of hypotheses
in prospective studies of patients. Our main aim is to promote and
integrate high quality research from bench to bedside in the field of
heart failure.
The CHFR Annual Symposium is a yearly event where ongoing scientific
work at the Center for Heart Failure Research is presented. We invite
national and international speakers to present their recent results. The
aim is to establish new collaborations within the CHFR and with national
and international researchers outside the CHFR.
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PROGRAM
08.00
08.20
Registration
Welcome by Geir Christensen (UUH)
Scientific session I: Myocardial function in heart failure
Chairmen: Ole M. Sejersted (UUH) and Halfdan Ihlen (RUH)
08.30
David Eisner, University of Manchester, United Kingdom
Calcium and the heart: in health and disease
09.00
Gerd Hasenfuss, University of Göttingen, Germany
Calcium cycling in heart failure
09.30
Øyvind Ellingsen, Norwegian University of Science and
Technology, Trondheim, Norway
Exercise-induced hypertrophy and reverse remodeling: from genes to
patients
10.00
Moderated poster session I: Inflammatory mediators in heart
failure
Moderators: Torbjørn Omland, Akershus University Hospital and Theis
Tønnessen (UUH)
Moderated poster session II: Receptor-mediated signaling and
novel therapies of heart failure
Moderators: Jan-Bjørn Osnes (UoO) and Reidar Bjørnerheim (UUH)
Moderated poster sessions III: Cardiac and skeletal muscle
dysfunction in heart failure
Moderators: Ivar Sjaastad (UUH) and Geir Øystein Andersen (UUH)
Moderated poster sessions IV: Cardiac and skeletal muscle
dysfunction in heart failure
Moderator: Jostein Hallén (Norwegian School of Sport Sciences)
11.30
Lunch
Scientific session II: Inflammation in heart failure
Chairmen: Pål Aukrust (RUH) and Lars Gullestad (RUH)
12.30
Douglas Mann, Baylor College of Medicine, Houston, USA
Immunmodulation in heart failure: past, present and future
13.15
Jan Kristian Damås, Rikshospitalet University Hospital, Oslo,
Norway
Chemokines as targets for treatment in heart failure
13.45
Break
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Scientific session III: Receptor-mediated signaling and novel therapies
of heart failure
Chairmen: Håvard Attramadal (RUH) and Arne Westheim (UUH)
14.05
Walter J. Koch, Jefferson Medical College, Philadelphia, USA
GRK2 as a potential therapeutic target and biomarker for heart failure
14.50
Break
15.00
Pieter Doevendans, University Medical Center Utrecht, the
Netherlands
Human cardiac progenitor cells for myocardial repair
15.30
Kenneth Dickstein, University of Bergen, Norway
Ongoing clinical trials in heart failure with drugs and devices
16.00
Break
Scientific session IV: Diagnostic strategies in heart failure
Chairman: Dan Atar, Aker University Hospital
16.20
Peter Buser, University Hospital Basel, Switzerland
The contribution of cardiac magnetic resonance imaging in the
evaluation of patients with heart failure: state of the art and future
developments
16.50
End of symposium
UUH
RUH
UoO
Ullevål University Hospital
Rikshospitalet University Hospital
University of Oslo
5
6
ABSTRACTS
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Moderated poster session I: Inflammatory mediators in
heart failure
P01
TNF induces anemia in mice with ischemic heart failure,
while NO represses erythropoiesis in non-ischemic heart failure
Per O Iversen, Kristin B Andersson, Alexandra V Finsen, Ivar Sjaastad, Thomas
von Lueder, Ole M Sejersted, Håvard Attramadal, Geir Christensen
P02
Myocardial expression of the TNF superfamily cytokine
LIGHT and its receptors in experimental and clinical heart failure
Christen P Dahl, Lars Gullestad, Erik Øie, Jan Kristian Damås, Pål Aukrust, Arne
Yndestad
P03
Elevated levels of activin A and follistatin in clinical and
experimental pulmonary arterial hypertension.
Arne Yndestad, Karl-Otto Larsen, Erik Øie, Thor Ueland, Camilla Smith, Bente
Halvorsen, Ivar Sjaastad, Ole Henning Skjønsberg, Turid M. Pedersen, OleGunnar Anfinsen, Jan Kristian Damås, Geir Christensen, Pål Aukrust, Arne K.
Andreassen
P04
Leukaemia inhibitory factor stimulates glucose transport
in isolated cardiomyocytes and induces insulin resistance after
chronic exposure
Geir Florholmen, G. Hege Thoresen, Arild C Rustan, Jørgen Jensen, Geir
Christensen, Vigdis Aas
P05
Connective tissue growth factor inhibits myocardial
growth, stimulates fibrosis, but preserves myocardial function in
chronic pressure overload
M. Shakil Ahmed, Thomas von Lueder, Jørgen A. Gravning, Thor Edvardsen, Erik
Øie, Birthe Mikkelsen, Otto A.Smiseth, Håvard Attramadal
P06
Novel cardioprotective role of connective tissue growth
factor in ischemia/reperfusion injury and heart failure
Jørgen A. Gravning, M. Shakil Ahmed, Vladimir Martinov, Thomas G. von Lueder,
Gabor Czibik, Thor Edvardsen, Birthe V. Mikkelsen, Otto A. Smiseth, Guro Valen,
Håvard Attramadal
P07
Plasma chromogranin A levels in the subacute phase are
predictive of exercise performance after myocardial infarction
Helge Røsjø, Kenneth Dickstein, Torbjørn Omland
8
P01
TNF induces anemia in mice with ischemic heart failure,
while NO represses erythropoiesis in non-ischemic heart failure
Per O Iversen1, Kristin B Andersson2, Alexandra V Finsen2, Ivar Sjaastad2,3,
Thomas von Lueder4, Ole M Sejersted2, Håvard Attramadal4, Geir Christensen2
Department of Nutrition, IMB, University of Oslo
Institute for Experimental Medical Research, Ullevaal University Hospital, Oslo
3
Department of Cardiology, Ullevaal University Hospital, Oslo
4
Institute of Surgical Research, Rikshospitalet-Radiumhospitalet Medical Center,
Oslo
1
2
Background: Anemia is an independent predictor of poor outcome in patients with
congestive heart failure (CHF). Insufficient erythropoietin production due to
impaired renal function cannot fully explain CHF-related anemia. In myocardial
infarction (MI), the most common cause of CHF, it is thought that a low-grade
inflammatory response induced by the infarction itself might inhibit blood cell
formation.
Aims: We wanted to examine (i) whether anemia would develop in mouse models
of non-ischemic CHF, and (ii) to identify possible inhibitors of bone marrow
hematopoiesis in CHF.
Methods: We examined hematopoiesis in adult mice (n=6-9 per group) with CHF
induced by MI (ischemia), and in two genetic mouse models of CHF induced by
altered sarcoplasmic reticulum (SR) function (non-ischemia), namely cardiacrestricted overexpression of the Ca2+-binding protein calsequestrin (CSQ), or
cardiac-restricted knockout of the SR Ca2+-ATPase 2 gene (SERCA2 KO).
Hematopoiesis was analyzed with colony assays.
Results: In control mice Hb was 14.1±0.1 g/dl, while it was reduced to 10.1±0.1;
9.7±0.2 and 9.6±0.1 g/dl in MI, CSQ and SERCA2 KO, respectively (P<0.05 for
all groups; mean±SEM). Colony numbers of immature hematopoietic progenitors
in the three CHF groups were reduced to 33±4 (MI), 39±2 (CSQ) and 32±4
(SERCA2 KO) (P<0.05 for all groups) compared with 71±5 in controls. In MImice, plasma TNF and TNF mRNA levels among bone marrow T cells increased
2-4 fold, and anti-TNF antibodies fully restored growth of those cells. However,
no such changes were observed in CSQ or SERCA2 KO compared with control
mice. In contrast, inhibition of hematopoiesis could be nearly completely
abolished with eNOS-blockade in CSQ and SERCA2 KO, but not in MI-mice.
Conclusions: This study demonstrates different mechanisms of anemia in CHF;
whereas TNF can impair hematopoiesis in MI-induced CHF, NO apparently is a
negative regulator of blood cell formation in non-ischemic CHF.
9
P02
Myocardial expression of the TNF superfamily cytokine
LIGHT and its receptors in experimental and clinical heart failure
Christen P Dahl, Lars Gullestad, Erik Øie, Jan Kristian Damås, Pål Aukrust, Arne
Yndestad
Institutt for indremedisinsk forskning, Rikshospitalet
Purpose: LIGHT (TNFSF14) is an inflammatory cytokine in the tumor necrosis
factor (TNF) superfamily that is involved in innate and adaptive immune
responses as well as in regulation of cell survival and proliferation. We have
previously shown that patients with heart failure (HF) have elevated gene
expression of LIGHT in peripheral blood mononuclear cells. In the present study,
we examined the myocardial expression of LIGHT and its receptors lymphotoxin-receptor (LTR) and herpes virus entry mediator (HVEM) in experimental and
clinical HF.
Methods and results: Myocardial gene expression of LIGHT, LTR, and HVEM was
analysed by real-time RT-PCR in a rat model of post-infarction HF 2, 7, and 28
days after induction of myocardial infarction (MI). LIGHT mRNA levels were
markedly elevated in the infarcted area and modestly increased in the nonischemic part of left ventricle throughout the study period. These changes in
LIGHT were accompanied by increased expression of its corresponding receptors
of which LTR was moderately increased at all time points after MI, while HVEM
gene expression reached the highest levels 28 days post-MI, representing a
chronic stage of HF. Immunohistochemical analysis of left ventricular tissue from
explanted failing human hearts demonstrated fairly strong LIGHT and HVEM
immunoreactivity in cardiomyocytes, endothelial cells, and vascular smooth
muscle cells, whereas weaker LTR immunoreactivity was observed in
cardiomyocytes.
Conclusion: Based on the important role of LIGHT in regulation of the immune
response, our findings in both clinical and experimental HF, suggest a role for
myocardial autocrine/paracrine LIGHT signalling pathways in the pathogenesis of
HF.
10
P03
Elevated levels of activin A and follistatin in clinical and
experimental pulmonary arterial hypertension.
Arne Yndestad1, Karl-Otto Larsen2,3,4, Erik Øie5,6, Thor Ueland1,7, Camilla Smith1,
Bente Halvorsen1, Ivar Sjaastad2,4,8, Ole Henning Skjønsberg3, Turid M.
Pedersen1, Ole-Gunnar Anfinsen9, Jan Kristian Damås1,10, Geir Christensen2,4, Pål
Aukrust1,10, Arne K. Andreassen4,9
Research Institute for Internal Medicine, Rikshospitalet University Hospital
Institute for Experimental Medical Research, Ullevål University Hospital
3
Department of Pulmonary Medicine, Ullevål University Hospital
4
Center for Heart Failure Research
5
Institute for Surgical Research, Rikshospitalet University Hospital
6
Medical Department, Diakonhjemmet Hospital
7
Department of Endocrinology, Rikshospitalet University Hospital
8
Department of Cardiology, Ullevål University Hospital
9
Department of Cardiology
10
Section of Clinical Immunology and Infectious Diseases, Rikshospitalet
University Hospital, University of Oslo, Norway.
1
2
Objectives. We sought to explore a role for activin A in pulmonary arterial
hypertension (PAH).
Background. Growing evidence links the transforming growth factor-beta
superfamily to the pathogenesis of PAH. Activin A, a member of this cytokine
family, is involved in regulation of cell proliferation, tissue remodeling and
inflammation.
Methods. Activin A and its binding protein follistatin were measured by enzyme
immune assays in mixed venous and arterial blood from patients with PAH
[idiopathic PAH (n=18), associated PAH (n=18), and chronic thromboembolic PH
(n=14)] and control subjects (n=14) and further explored in experimental
studies.
Results. i) Levels of activin A and follistatin were significantly elevated in patients
with PAH. ii) Activin A levels were significantly related to mortality in the patient
cohort. iii) PAH patients had significantly higher follistatin levels in the femoral
than in the pulmonary artery indicating net release of follistatin from lungs. iv)
Lungs from mice with hypoxia-induced PAH exhibited increased gene expression
of activin A and follistatin, and also elevated levels of phosphorylated and total
Smad2. v) Immunohistochemistry of human lung autopsies from PAH patients
and lungs with experimental PAH localized activin A primarily to alveolar
macrophages and bronchial epithelial cells; vi) Activin A induced gene expression
of endothelin-1 and plasminogen activator inhibitor (PAI)-1 in endothelial cells
and vascular smooth muscle cell, respectively; mediators that both could
contribute to vascular remodeling.
Conclusion. Our findings in both clinical and experimental studies suggest a role
for activin A in the development of PAH.
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P04
Leukaemia inhibitory factor stimulates glucose transport
in isolated cardiomyocytes and induces insulin resistance after
chronic exposure
Geir Florholmen1,2, G. Hege Thoresen3, Arild C Rustan3, Jørgen Jensen4, Geir
Christensen1,2, Vigdis Aas3,5
1
2
3
4
Institute for Experimental Medical Research, Ullevaal University Hospital.
Center for Heart Failure Research, University of Oslo.
Department of Pharmaceutical Biosciences, School of Pharmacy, University of
Oslo.
National Institute of Occupational Health, Oslo. (5)Faculty of Health Sciences,
Oslo University College.
Hypertrophic and failing hearts have increased utilization of glucose, but develop
insulin resistance and reduced ATP production. Increased levels of the
interleukin-6-related cytokine leukaemia inhibitory factor (LIF) are found in failing
hearts, and we have recently shown that LIF reduces ATP production in isolated
cardiomyocytes. In the present study we investigated effects of LIF on glucose
metabolism, and how LIF-treated cells respond to insulin stimulation.
Cardiomyocytes were isolated from adult Wistar rats and treated with 1 nmol/l
LIF. Acute LIF treatment increased deoxyglucose uptake compared to control, but
no additive effect was observed in cardiomyocytes treated with both LIF and
insulin. The phosphatidylinositol 3-kinase inhibitor wortmannin did not affect the
LIF-induced glucose uptake. LIF had no effect on AMPK phosphorylation.
Cardiomyocytes treated with LIF for 48h did not respond to insulin by increasing
deoxyglucose uptake and showed reduced insulin-mediated uptake of oleic acid
and formation of complex lipids compared to control cells. Chronic LIF treatment
increased gene expression of the suppressor of cytokine signalling (SOCS) 3 and
reduced expression of the glucose transporter GLUT4. In line with these
observations, chronic LIF treatment reduced insulin-mediated phosphorylation of
both Akt/PKB and glycogen synthase kinase (GSK)-3. Acute LIF treatment
increased glucose uptake in isolated cardiomyocytes by another pathway than
insulin. Chronic LIF treatment induced insulin resistance, possibly mediated by
altered expression of SOCS3 and GLUT4, and impaired insulin-mediated
phosphorylation of GSK-3 and Akt/PKB.
12
P05
Connective tissue growth factor inhibits myocardial
growth, stimulates fibrosis, but preserves myocardial function in
chronic pressure overload
M. Shakil Ahmed1, Thomas von Lueder1,2, Jørgen A. Gravning1, Thor Edvardsen1,2,
Erik Øie1,2, Birthe Mikkelsen1, Otto A.Smiseth1,2, Håvard Attramadal1,2
1
2
Inst. for Surgical Research and
Dept. of Cardiology, Rikshospitalet-Radiumhospitalet Medical Center and
University of Oslo.
Background: Myocardial expression of connective tissue growth factor (CTGF) is
dramatically induced in heart failure (HF) of diverse etiologies. However, the
physiologic and pathophysiologic roles of myocardial CTGF remain unresolved.
Methods and Results: To elucidate the actions of myocardial CTGF and its
putative role in HF transgenic mice with cardiac-restricted overexpression of
CTGF were generated. Transgenic CTGF (Tg-CTGF) mice had slightly lower
cardiac mass than that of non-transgenic littermate controls (NLC) (heart
weight/tibia length of 4 months old male Tg-CTGF vs. NLC; 58.9±2.7 vs.
68.1±1.2 mg/cm, p<0.05). Consistently, echocardiography revealed significantly
smaller end-diastolic interventricular septum thickness in Tg-CTGF. Simultaneous
LV pressure-volume analysis did not disclose significant alterations of contractility
and cardiac output. Histochemical analysis of myocardial tissue revealed
interstitial fibrosis in Tg-CTGF mice. Increased myocardial collagen was confirmed
by quantitation of myocardial hydroxyproline contents by HPLC (1.10±0.03
vs.0.82±0.05 pmol/mg dry weight in Tg-CTGF vs. NLC mice; p<0.05).
Consistently, increased myocardial procollagen--I and -III mRNA levels in TgCTGF vs. NLC hearts were found. Morover, increased expression of
antihypertrophic TGF-2 and GDF-15 mRNA, and decreased expression of EGF
mRNA in Tg-CTGF vs. NLC mice was detected. Tg-CTGF and NLC mice were
subjected to pressure overload by abdominal aortic banding (AB) or shamoperation (SH). Four weeks after AB, significant elevations of cardiac mass were
observed both in Tg-CTGF-AB and NLC-AB mice. However, cardiac hypertrophy
was significantly diminished in Tg-CTGF-AB versus NLC-AB. Simultaneous PVanalysis provided evidence of cardiac dysfunction in NLC-AB mice, i.e.
significantly increased LVEDD, LVEDP, and decreased stroke volume and cardiac
output compared to NLC-SH mice. Strikingly, Tg-CTGF-AB revealed essentially
preserved LV pressure-volume relations. Elevations of myocardial BNP mRNA
expression were significantly attenuated in Tg-CTGF-AB compared to NLC-AB
mice.
Conclusion: Myocardial CTGF causes interstitial fibrosis, but no evidence of
restrictive cardiac dysfunction, and preserves left ventricular function due to
pressure overload.
13
P06
Novel cardioprotective role of connective tissue growth
factor in ischemia/reperfusion injury and heart failure
Jørgen A. Gravning1, M. Shakil Ahmed1, Vladimir Martinov2, Thomas G. von
Lueder1, 3, Gabor Czibik2, Thor Edvardsen1,3, Birthe V. Mikkelsen1, Otto A.
Smiseth1, 3, Guro Valen2, Håvard Attramadal1, 3
1
2
3
Inst. for Surgical Research and
Dept. of Physiology, University of Oslo
Dept. of Cardiology, Rikshospitalet-Radiumhospitalet Medical Center, Oslo,
Norway
Background: The myocardial growth factor CCN2/CTGF - connective tissue growth
factor has been shown to be robustly induced in experimental models of heart
failure (HF) as well as in human HF. Yet, its role in the pathophysiologic
mechanisms of HF is unknown.
Methods and Results: To elucidate the role of myocardial CTGF in
ischemia/reperfusion injury and HF, transgenic mice with cardiac-restricted
overexpression of CTGF were employed. Transgenic CTGF (Tg-CTGF) hearts had
slightly lower cardiac mass than that of non-transgenic littermate controls (NLC)
(heart weight/tibia length of 4 months old male Tg-CTGF vs. NLC mice; 58.9±2.7
vs. 68.1±1.2 mg/cm, p<0.05). However, cardiac function assessed by LV
pressure-volume analysis was essentially unaltered. Global analysis of gene
expression by DNA microarray revealed activation of distinct gene programs in
Tg-CTGF hearts, including induction of endoplasmatic reticulum stress response
genes, antiapoptotic factors, and ECM proteins, suggesting involvement of CTGF
in tissue protection. Also, several signalling mediators reported to confer
cardioprotection, e.g. GDF-15, Toll-like receptor-4, and HIF-1, were elevated in
Tg-CTGF vs. NLC hearts. Langendorff-perfused hearts from Tg-CTGF (n=9) and
NLC mice (n=9) were subjected to 40 min of global (no-flow) ischemia and
subsequently 60 min of reperfusion. End-point analysis demonstrated markedly
diminished infarct size in Tg-CTGF vs. NLC hearts (21.0±3.9 vs. 49.1±6.3 %,
p<0.01) and enhanced recovery of LV function (LV developed pressure
81.3±18.6 vs. 40.4±21.7 mmHg, p<0.01). Western blot analysis also revealed
increased levels of phosphorylated (activated) Akt/PKB and GSK-3 in Tg-CTGF
vs. NLC hearts, demonstrating activation of bona fide cardioprotective signalling
pathways. Consistently, Tg-CTGF mice subjected to pressure-overload by
abdominal aortic banding displayed less hypertrophy and preserved LV pressurevolume relations compared with banded NLC mice.
Conclusion: These data provide novel evidence that CTGF confers
cardioprotection by preemptive preconditioning due to reprogramming of gene
expression and may act as endogenous survival factor in HF.
14
P07
Plasma chromogranin A levels in the subacute phase are
predictive of exercise performance after myocardial infarction
Helge Røsjø1, Kenneth Dickstein2, Torbjørn Omland1
1
2
Akershus University Hospital, Lørenskog, Norway
Stavanger University Hospital, Stavanger, Norway
Introduction: Acute myocardial infarction (AMI) is associated with complex
neuroendocrine activation and varying degrees of subsequent functional
impairment. Chromogranin A (CgA) is widely distributed throughout the
neuroendocrine system and may, due to its long in vivo and in vitro half-life, be
an attractive candidate for assessment of neuroendocrine activity. Recently,
plasma levels of CgA have been related to the severity of symptoms and
prognosis in patients with chronic heart failure.
Hypothesis: We hypothesized that CgA levels in the subacute phase would be
predictive of exercise performance after AMI and perform better or equally well
as traditional neurohormonal markers.
Methods: Blood for determination of neurohormonal markers were obtained on
day 3 after symptom debut in 73 male patients (median age 69, interquartile
range 66-75 yrs) with documented AMI. CgA in plasma was determined by
radioimmunoassay, norepinephrine with HPLC and electrochemical detection and
BNP with an immunoradiometric method (ShionogiRIA). Functional capacity 6
months post AMI was assessed during symptom-limited exercise on an upright
bicycle ergometer, using a continuous ramp protocol with 20 Watt increments
per min and expressed as exercise duration. Patients were prospectively
subdivided according to the presence or absence of significant ST-segment
depression.
Results: The correlations between neuroendocrine markers and exercise duration
are shown in the table.
Conclusion: Plasma CgA obtained in the subacute phase after AMI are predictive
of subsequent exercise performance and performs at least as well as tradional
neuroendocrine markers. The relationship is particularly pronounced in patients
without exercise-induced ischemia.
Table. Correlations between neuroendocrine markers and exercise duration
All patients
No ST
ST depression
depression
Chromogranin A
r= –0.39 *
r= –0.58 *
r= –0.33 †
BNP
r= –0.41 *
r= –0.46 *
r= –0.35 †
Norepinephrine
r= –0.20
r= –0.40 †
r= 0.008
* P < 0.01, † P < 0.05
15
Moderated poster sessions II: Receptor-mediated signaling
and novel therapies of heart failure
P08
Atorvastatin normalizes endothelial function in healthy
smokers
Stefan Agewall
P09
Inhibition of G protein-coupled receptor kinase-3 (GRK3)
rescues cardiac dysfunction after pressure overload
Thomas G. von Lueder, Jørgen A. Gravning, Thor Edvardsen, Ole Jacob How,
Ellen Aasum, Leif E. Vinge, M. Shakil Ahmed, Birthe V. Mikkelsen, Terje S.
Larsen, Otto A. Smiseth, Håvard Attramadal
P10
The 5-HT4 receptor – a foetal gene reactivated in heart
failure?
Trond Brattelid, Silje V.S. Bekkevold, Eirik Qvigstad, Lise Román Moltzau, Dagny
L. Sandnes, Jon Arne K. Birkeland, Ivar Sjaastad, Finn Olav Levy
P11
5-HT4 mediated positive inotropic response is regulated
by PDE3 in failing rat heart and human ventricle
Faraz Afzal, Kjetil W. Andressen, Halvor K. Mørk, Magnus Aronsen, Ivar Sjaastad,
Lise R. Moltzau, Tor Skomedal, Finn Olav Levy, Jan-Bjørn Osnes, Eirik Qvigstad
P12
Effects of treatment with a 5-HT4 serotonin receptor
antagonist in heart failure
Jon Arne Kro Birkeland, Ivar Sjaastad, Trond Brattelid, Eirik Qvigstad, Enrica Ratti
Moberg, Kurt A. Krobert, Reidar Bjørnerheim, Tor Skomedal, Ole M. Sejersted,
Jan-Bjørn Osnes, Finn Olav Levy
P13
Neurohormonal signalling during decompensation in
SERCA knock out mice.
Karina Hougen, Jan Magnus Aronsen, Jon Arne Birkeland, Eirik Qvigstad, Bill
Louch, Kristin Andersson, Geir Christensen, Ole Sejersted, Finn Olav Levy, Ivar
Sjaastad
P14
Muscarinic receptor activation elicits an inotropic effect in
failing rat ventricle through myosin light chain phosphorylation
Rizwan I. Hussain, Eirik Qvigstad, Jon Arne Birkeland, Ivar Sjaastad, Annette
Glende, Hilde Eikemo, Trond Brattelid, Jan-Bjørn Osnes, Tor Skomedal, Finn Olav
Levy, Kurt A. Krobert
P15
Evidence for a novel kinase phosphorylating MLC-2 in
quiescent cardiomyocytes
Hilde Eikemo, Finn Olav Levy, Tor Skomedal, Jan-Bjørn Osnes
16
P08
Atorvastatin normalizes endothelial function in healthy
smokers
Stefan Agewall1
1
Division of Cardiology, Aker University Hospital
Endothelial function predicts cardiovascular disease. The aim of this study was to
examine whether endothelial function in smokers with normal cholesterol levels
could be normalized by treatment with 80 mg atorvastatin in comparison with a
control group. Healthy smokers (n=20) and non-smokers (n=20) were
randomized to 80 mg atorvastatin or to placebo in an open, randomized crossover study. Endothelial functional (Flow-mediated dilatation; FMD) was
measured. At baseline smokers had a lower FMD compared to the non-smoking
group, 2.2 % ± 0.5 and 4.5 % ± 0.9, respectively (p<0.05). In the smoker
group FMD increased to 4.0 % ± 0.8 during treatment with atorvastatin (p<0.05)
and returned to basal level during placebo, 2.3 % ± 0.6. In the non-smoking
group FMD was unaffected by both atorvastatin and placebo. The net change of
total cholesterol or LDL cholesterol was not associated with the net change of
FMD when the group was considered as a whole or the smoking group was
considered separately. In conclusion, an improved endothelial function was
observed in a group of smokers, when they were on 80 mg atorvastatin, whereas
atorvastatin had no effect on endothelial function in the non-smoking group. The
improved FMD among smokers was independent of the LDL decrease during
atorvastatin treatment. The results show that poor endothelial function in
smokers with normal lipid levels can be improved by a statin treatment.
17
P09
Inhibition of G protein-coupled receptor kinase-3 (GRK3)
rescues cardiac dysfunction after pressure overload
Thomas G. von Lueder1, 2, Jørgen A. Gravning1, Thor Edvardsen1, 2, Ole Jacob
How3, Ellen Aasum3, Leif E. Vinge1, M. Shakil Ahmed1, Birthe V. Mikkelsen1, Terje
S. Larsen3, Otto A. Smiseth1, 2, Håvard Attramadal1, 2
1
2
3
Inst. for Surgical Research and
Dept. of Cardiology, Rikshospitalet-Radiumhospitalet Medical Center and
University of Oslo, and
Dept. of Physiology, University of Tromsø, Norway
Background: Previous studies of transgenic mice with cardiac-restricted inhibition
of GRK3 disclosed increased cardiac performance and phenotype consistent with
1-adrenergic receptor hyperresponsiveness. This is in contrast to GRK2 (ARK1), an isoenzyme with selectivity for -adrenergic receptors. The aim of this
study was to investigate the role of GRK3 during evolving cardiac dysfunction
after pressure overload.
Methods and results: GRK3ct transgenic mice (Tg-GRK3ct) with cardiac-restricted
expression of mini-gene encoding the carboxyl-terminal membrane targeting
domain of GRK3 were employed. Inhibition of endogenous GRK3 was confirmed
by enhanced 1-AR mediated activation of ERK1/2 in cardiac myocytes from TgGRK3ct as compared to non-transgenic littermate controls (NLC). Tg-GRK3ct
mice characteristically displayed myocardial hypercontractility despite unaltered
cardiac mass compared to NLC. Pressure overload was induced by abdominal
aortic banding (AB) in weight-matched male Tg-GRK3ct (GRK3ct-AB) and NLC
(NLC-AB) mice and compared with sham-operated Tg-GRK3ct (GRK3ct-SH) and
NLC (NLC-SH) mice. Echocardiography revealed similar baseline cardiac
dimensions among all groups. Eight and twelve weeks after AB, significant
increases of cardiac mass were found in AB mice compared to sham, but no
differences between GRK3ct-AB and NLC-AB were discerned. Eight weeks after
AB, analysis of electrically paced, ex vivo perfused hearts at increasing filling
pressures revealed preserved end-diastolic and end-systolic pressure-volume
(PV) relations in Tg-GRK3ct-AB (n=10), with substantially higher cardiac output
and dP/dtmax compared to NLC-AB (n=10). Twelve weeks after AB, simultaneous
in vivo PV-analysis revealed elevated end-diastolic pressure (10.6±3.1 vs.
2.7±2.2 mmHg, p<0.05) and lower cardiac output (7379±738 vs. 9847±785
μL/min, p<0.05) in NLC-AB (n=11) compared to GRK3ct-AB (n=16).
Consistently, myocardial mRNA levels of B-type natriuretic peptide were
substantially elevated in NLC-AB vs. GRK3ct-AB (p<0.05).
Conclusion: Inhibition of cardiac GRK3 in mice rescues cardiac dysfunction and
heart failure after pressure overload.
18
P10
The 5-HT4 receptor – a foetal gene reactivated in heart
failure?
Trond Brattelid1,2, Silje V.S. Bekkevold1,2, Eirik Qvigstad1,2, Lise Román Moltzau1,2,
Dagny L. Sandnes1, Jon Arne K. Birkeland2,3, Ivar Sjaastad2,3,4, Finn Olav Levy1,2
1
2
3
4
Department of Pharmacology, University of Oslo, Oslo, Norway
Center for Heart Failure Research, University of Oslo, Oslo, Norway
Institute for Experimental Medical Research, University of Oslo, Oslo, Norway
Department of Cardiology, Ullevaal University Hospital, Oslo, Norway
A positive inotropic responsiveness to serotonin, mediated by 5-HT4 and 5-HT2A
receptors, appears in the ventricle of rats with post-infarction congestive heart
failure (CHF). A hallmark of CHF is a transition towards a foetal genotype which
correlates with loss of cardiac functions. To explore the role of 5-HT4, 5-HT2A and
5-HT2B receptors in the foetal heart, ventricle from Wistar rats were collected 3
and 1 days before expected birth (days -3 and -1), as well as 1, 3, 5 and 113
days after birth, and examined for expression of 5-HT4, 5-HT2A and 5-HT2B
serotonin receptor mRNA (real-time quantitative RT-PCR) as well as contractile
function mediated by these receptors ex vivo.
Both 5-HT4 mRNA expression and function was highest at day -3 and decreased
gradually from day -3 to day 5, with a further decrease to adult levels (day 113).
The levels increased in CHF, but the levels seen in the foetal ventricle were higher
than the levels in CHF. The 5-HT2A and 5-HT2B receptor mRNA levels increased to
a maximum immediately after birth, a situation associated with an acute increase
in wall stress, but only the 5-HT2A receptor mediated a ventricular inotropic
response.
These results indicate that the 5-HT4 receptor is a representative of the foetal
cardiac gene program and might play a significant role in early cardiac
development and CHF. The 5-HT2A and 5-HT2B receptors might therefore play
important roles in postnatal heart development associated with an acute increase
in wall stress and a subsequent hypertrophy.
19
P11
5-HT4 mediated positive inotropic response is regulated
by PDE3 in failing rat heart and human ventricle
Faraz Afzal1, Kjetil W. Andressen1, Halvor K. Mørk2, Magnus Aronsen2, Ivar
Sjaastad2,3, Lise R. Moltzau1, Tor Skomedal1, Finn Olav Levy1, Jan-Bjørn Osnes1,
Eirik Qvigstad1.
1
2
3
4
Department of Pharmacology, University of Oslo
Institute for Experimental Medical Research, Ullevål University Hospital
Department of Cardiology, Ullevål University Hospital
Center for Heart Failure Research, University of Oslo
Background: In chronic heart failure, the left ventricle becomes sensitive to
serotonin (5-HT) due to the appearance of functional Gs-coupled 5-HT4 receptors.
Phosphodiesterase (PDE) 3 and 4 account for >90% of the total cardiac cAMP
PDE activity and PDE 4 primarily couples to the cAMP pool activated by adrenoceptors.
Objective: Explore the involvement of phosphodiesterases in the regulation of
ventricular 5-HT4 mediated positive inotropic effect (PIE) induced in failing rat
and human heart.
Results: In rat papillary muscles 5-HT4 stimulation exerted a PIE accompanied by
an increase in total cAMP and phospholamban phosphorylation. The PIE was
increased by a non-selective PDE inhibitor (IBMX, 10µM) and suppressed by the
PKA inhibitor H89 (20µM) demonstrating the involvement of the cAMP-PKA
pathway. The PDE4 inhibitor rolipram (10µM) did not significantly increase the 5HT4 response. The PDE3 inhibitor cilostamide (1µM) increased the PIE of
serotonin without changing the sensitivity. Combined PDE3/4 inhibition further
enhanced the PIE and increased the sensitivity to 5-HT. In failing human ventricle
PDE3 inhibition significantly increased the PIE whereas PDE4 inhibition had no
significant effect.
Conclusions: The inotropic response to 5-HT4 is mediated through a cAMP-PKA
dependent pathway. PDE3 is the main regulator of this response and the
involvement of PDE4 is demasked by inhibition of PDE3.
20
P12
Effects of treatment with a 5-HT4 serotonin receptor
antagonist in heart failure
Jon Arne Kro Birkeland1, 2, Ivar Sjaastad1, 2, 4, Trond Brattelid1, 3, Eirik Qvigstad1,3,
Enrica Ratti Moberg1, 3, Kurt A. Krobert1, 3, Reidar Bjørnerheim1, 4, Tor Skomedal1,
3
, Ole M. Sejersted1, 2, Jan-Bjørn Osnes1, 3, Finn Olav Levy1, 3
1
2
3
4
Center for Heart Failure Research, University of Oslo
Institute for Experimental Medical Research, Ullevaal University Hospital
Department of Pharmacology, University of Oslo
Department of Cardiology, Ullevaal University Hospital
We have previously found that a positive inotropic response (PIR) to serotonin (5HT) appears in the left ventricle (LV) of rats with congestive heart failure (CHF),
associated with upregulation of the Gs-coupled 5-HT4 receptor. We hypothesized
that in CHF, 5-HT4 receptor stimulation could be of disadvantage, as stimulation
of beta-adrenoceptors. Therefore, to investigate whether chronic 5-HT4 receptor
blockade improves cardiac function in CHF, rats were randomized to receive the
5-HT4 antagonist SB207266 (0.5 mg/kg/24h; MIint; n=30) or placebo (MIpl;
n=23) through mini-osmotic pumps for 6 weeks subsequent to induction of
postinfarction CHF. LV diastolic function improved, as assessed by a 4.6% lower
LV diastolic diameter and 24.2% lower mitral flow deceleration in MIint compared
to MIpl (both p<0.05). SB207266 reduced LV systolic diameter by 6.1%, heart
weight by 10.2% and lung weight by 13.1% (all p<0.05). The changes in
posterior wall thickening and shortening velocity, cardiac output, LV systolic
pressure and (dP/dt)max, parameters of LV systolic function, did not reach
statistical significance. In isolated papillary muscles, the PIR to isoproterenol (10
µM) increased by 36% and the response to 5-HT (10 µM) decreased by 57% in
MIint compared to MIpl (both p<0.05). Real-time quantitative RT-PCR revealed a
non-significant reduction in ANP, 5-HT4(b), 5-HT2A and MHCβ mRNA levels and
MHCβ/MHC-ratio, and a non-significant increase in MCH mRNA level in MIint
compared to MIpl. We conclude that treatment with the 5-HT4 antagonist
SB207266 to some extent improved in vivo cardiac function and ex vivo
myocardial function, suggesting a possible beneficial effect of treatment with a 5HT4 receptor antagonist in CHF. Since functional 5-HT4 receptors also exist in
failing human ventricle, this may be relevant for human heart failure.
21
P13
Neurohormonal signalling during decompensation in
SERCA knockout mice
Karina Hougen1,3, Jan Magnus Aronsen1,3, Jon Arne Birkeland1,3, Eirik Qvigstad2,3,
Bill Louch1,3, Kristin B. Andersson1,3, Geir Christensen1,3, Ole Sejersted1,3, Finn
Olav Levy2,3, Ivar Sjaastad1,3,4
1
2
3
4
Institute for Experimental Medical Research, University of Oslo
Department of Pharmacology, University of Oslo
Center for Heart Failure Research, University of Oslo
Department of Cardiology, Ullevaal University Hospital
Reduced sarcoplasmic reticulum (SR) function is a hallmark of heart failure. Four
weeks after deletion of the SR Ca2+ ATPase (SERCA) gene, mice only exhibit a
minor reduction of cardiac function in face of negligible myocardial levels of the
SERCA protein. However, these mice develop overt heart failure during the next 3
weeks, although no further reduction in SERCA protein levels are detected. We
wanted to examine adrenergic and serotonergic signalling in cardiac myocytes
during development of heart failure in these mice.
Cardiomyocytes isolated from both SERCA2 KO and control mice 4 and 7 weeks
after administration of tamoxifen, were field stimulated at 1Hz. Serotonin
receptor (5-HT4 and 5-HT2A) and adrenoceptor mediated effects on fractional cell
shortening (FS) was measured in the presence of appropriate receptor blockers
present. In a different set of experiments, contractions and Ca2+ transients (fluo4, expressed as F/F0) were measured simultaneously.
Fractional shortening in SERCA2 KO cardiomyocytes was only 9.0% of controls at
4 weeks, and 15.7% at 7 weeks (p=NS 4 vs. 7 weeks). In SERCA2 KO
cardiomyocytes serotonin increased FS with 56.2 and 88.2% at 4 and 7 weeks
respectively. In controls serotonin increased FS with 8.1% and 6.9% respectively.
Selective stimulation of 5-HT2A receptors increased FS with 46.9% and F/F0 with
14.9% in SERCA2 KO cardiomyocytes at 7 weeks. In controls FS increased 23.6%
and F/F0 increased 3.7%. Isoprenaline increased FS by 284% in SERCA2 KO
cardiomyocytes 7 weeks after administration of tamoxifen, compared to 90% in
control mice.
In conclusion, both serotonergic and adrenergic signaling pathways are functional
in SERCA2 KO cardiomyocytes, and are able to enhance cardiomyocyte
contractility in vitro.. These responses do not seem to be altered between 4 and 7
weeks after SERCA2 deletion. Thus, other mechanisms might be more important
for the observed reduction in vivo function from 4 to 7 weeks.
22
P14
Muscarinic receptor activation elicits an inotropic effect in
failing rat ventricle through myosin light chain phosphorylation
Rizwan I. Hussain1,2, Eirik Qvigstad1,2, Jon Arne Birkeland1,3, Ivar Sjaastad1,3,4,
Annette Glende1,2, Hilde Eikemo1,2, Trond Brattelid1,2, Jan-Bjørn Osnes1,2, Tor
Skomedal1,2, Finn Olav Levy1,2, Kurt A. Krobert1,2
1
2
3
4
Center for Heart Failure Research, Faculty of Medicine, University of Oslo
Department of Pharmacology, University of Oslo
Institute for Experimental Medical Research, Ullevaal University Hospital,
University of Oslo
Department of Cardiology, Heart and Lung Center, Ullevaal University Hospital
Muscarinic receptor mediated inhibition of cAMP dependent inotropic effects is
enhanced in papillary muscles from rats with congestive heart failure (CHF).
Muscarinic stimulation by the agonist carbachol (CCh) increases myofilament Ca2+
sensitivity without an inotropic effect in normal myocardium. Therefore, we
tested the hypothesis that muscarinic activation by CCh could elicit direct
inotropic effects in ventricular myocardium from rats with CHF through
enhancement of Ca2+ sensitization. Contractile force was measured in papillary
muscles from male Wistar rats having undergone ligation of the left coronary
artery (and exhibiting CHF) or sham surgery. CCh evoked a positive inotropic
effect (20% above basal) approximating 36% of that elicited by isoproterenol
(56% above basal), only in rats with CHF. In contrast, CCh elicits only a transient
negative inotropic effect in sham rats. The CCh mediated inotropic effect did not
correlate with infarction size but did correlate with increased left ventricular end
diastolic pressure, heart weight and lung weight, primary indicators of CHF
severity. The dose response curve for CCh was shifted to higher concentrations
only in the presence of the relatively specific M2 antagonist (AF-DX 116) (pD2values with AF-DX 5.68 ± 0.12 vs. 6.28 ± 0.10 without); whereas, the
antagonists nitrocaramiphen (M1) and 4-DAMP (M3/5) were without effect. The
CCh mediated inotropic effect was insensitive to pertussis toxin but was
attenuated by the myosin light chain kinase inhibitor ML-9 (8% above basal) and
by the Rho-kinase inhibitor Y-27632 (9% above basal). CCh increased
phosphorylation of myosin light chain that was also attenuated by ML-9 and Y27632. In conclusion, these data indicate that muscarinic receptor activation,
possibly M2, mediates an inotropic effect in failing ventricular myocardium, not
observed in normal rat heart. The muscarinic inotropic effect occurs through a
signaling pathway that enhances myosin light chain phosphorylation and thus,
likely Ca2+ sensitization.
23
P15
Evidence for a novel kinase phosphorylating MLC-2 in
quiescent cardiomyocytes
Hilde Eikemo1,2, Finn Olav Levy1,2, Tor Skomedal1,2, Jan-Bjørn Osnes1,2
1
2
Department of Pharmacology and
Center for heart failure research, University of Oslo, Norway
Myosin light chain 2 (MLC-2) is a 20 kDa protein located in the hinge region of
the heavy myosin molecule. MLC-2 in smooth muscle must be phosphorylated in
order to induce contraction. In cardiac muscle, MLC-2 phosphorylation facilitates
contraction and is assumed to mediate inotropic responses to various
neurohumoral stimuli. The aim of this study was to elucidate some properties of
kinases phosphorylating MLC-2 in quiescent cardiomyocytes. Cardiomyocytes
were isolated from adult rat hearts by collagenase perfusion and attached to
laminine. Phosphorylated and non-phosphorylated MLC-2 were separated using
charged gel electrophoresis, and were identified by anti-ventricular MLC-2
monoclonal antibody. The phosphorylation status of MLC-2 is reported as
phosphorylated MLC-2 as percent of total MLC-2. Data are presented as
mean±SEM. Calyculin A (myosin light chain phosphatase inhibitor) unmasked a
kinase activity by increasing MLC-2-phosphorylation dose-dependently from
15.9%±1.8% (0 M) to 45.9%±4.0% (10-7 M), p<0.001, n=6, with a log EC50 of 7.96±0.05, and time-dependently from 18.5%±1.3% at 0 min to 50.1%±3.3%
at 80 min, p<0.001, n=12, with half maximal phosphorylation at 15 min and
reaching a plateau at about 40 min after addition of calyculin A (10-7 M).
Preincubating (45 min) cardiomyocytes with ML-9 (6x10-5 M; myosin light chain
kinase (MLCK) and Rho kinase inhibitor) reduced basal MLC-2 phosphorylation,
but the effect was reversed by subsequent treatment (time-response curve) with
calyculin A (10-7 M). Preincubating with ML-7 (10-5 M; MLCK inhibitor) had no
effect, but staurosporine (10-5 M; a broad spectrum protein kinase inhibitor)
removed all kinase activity and rendered only a slight effect of calyculin A after
80 min treatment. Conclusion: In quiescent cardiomyocytes, MLC-2 is
phosphorylated by a staurosporine sensitive but ML-7 and ML-9 insensitive
kinase(s), different from MLCK, probably being Ca2+-independent.
24
25
Moderated poster session III: Cardiac and skeletal muscle
dysfunction in heart failure
P16
Heart function in adult cardiac Serca2null mice.
Kristin Brevik Andersson, Jon Arne Kro Birkeland, Alexandra Vanessa Finsen,
William E. Louch, Ivar Sjaastad, Yibin Wang, Ju Chen, Jeffery D. Molkentin,
Kenneth R. Chien, Ole M. Sejersted, Geir Christensen
P17
Increased transsarcolemmal Ca2+ flux in mice with
cardiomyocyte-specific deletion of the Serca2 gene
William E. Louch, Jon Arne Kro Birkeland, Alexandra Vanessa Finsen, Kristin
Brevik Andersson, Ivar Sjaastad, Yibin Wang, Ju Chen, Jeffery D. Molkentin,
Kenneth R. Chien, Ole M. Sejersted, Geir Christensen
P18
Role of Serca2 in the embryonic heart
Cecilie Sjåland, Geir Christensen, Kristin Brevik Andersson
P19
Interplay between the systolic Ca transient, SR Ca content
and Ito: a recipe for arrhythmias
K.M. Dibb, H.K. Graham, L.C. Diffley, J.D. Clarke, A.W. Trafford
P20
Reduced t-tubule density and less synchronous Ca2+
release in cardiomyocytes from rats with congestive heart failure
Fredrik Swift, William E. Louch, Jon Arne Kro Birkeland, Marion J. Thomas, Mark
B. Cannell, Ivar Sjaastad, Ole M. Sejersted
P21
Myocardial infarction in adult mice induces hypertrophy
and congestive heart failure without reduced contraction of noninfarcted cardiomyocytes.
Halvor K. Mørk, Ivar Sjaastad, Ole M. Sejersted, William E. Louch
P22
Differential regulation of genes encoding contractile
proteins during development of heart failure after myocardial
infarction in mice
Cathrine Husberg, Alexandra Finsen, Ståle Nygård, Geir Christensen
P23
Calcium handling in cardiomyoctes isolated from rats with
post infarction heart failure – effect of hypoxia/reoxygenation
and ouabain exposure.
Mohammad Nouri Sharikabad, Janne Pedersen, Ivar Sjaastad, Ole M. Sejersted,
Odd Brørs
P24
Increased amounts of PP1 and PP2A mediating reduced
serine 16 phosphorylation of phospholamban is a possible
mechanism for cardiac dysfunction in alveolar hypoxia
Karl-Otto Larsen, Birgitte Lygren, Ivar Sjaastad, Kurt A. Krobert, Geir Florholmen,
Kristin Arnkværn, Ole Henning Skjønsberg, Kjetil Taskén, Geir Christensen
26
P16
Heart function in adult cardiac Serca2null mice.
Kristin Brevik Andersson1,2, Jon Arne Kro Birkeland1,2, Alexandra Vanessa
Finsen1,2, William E. Louch1,2, Ivar Sjaastad1,2,3, Yibin Wang4, Ju Chen5, Jeffery D.
Molkentin6, Kenneth R. Chien7, Ole M. Sejersted1,2, Geir Christensen1,2
1
2
3
4
5
6
7
Institute for Experimental Medical Research, Ullevaal University Hospital
Center for Heart Failure Research, University of Oslo
Department of Cardiology, Heart and Lung Center, Ullevaal University Hospital
Departments of Anesthesiology, Physiology and Medicine, David Geffen School
of Medicine, University of California Los Angeles, Los Angeles, CA, USA.
Institute of Molecular Medicine, University of California San Diego, La Jolla,
California, USA.
Department of Pediatrics, University of Cincinnati, Children's Hospital Medical
Center, Division of Molecular Cardiovascular Biology, Cincinnati, Ohio, USA.
Cardiovascular Research Center, Massachusetts General Hospital, Charlestown,
Massachusetts, USA.
The generation of Ca2+ transients in cardiomyocytes is essential for heart
function. The SERCA2 pump maintains the sarcoplasmic reticulum (SR) Ca2+ store
by sequestration of Ca2+ from cytosol into the SR. Loss of SERCA2 in adult hearts
is therefore expected to cause immediate severe myocardial contractile
dysfunction and death. We have generated a new mouse model to test this
hypothesis.
Homozygous Serca2flox mice, were mated with Tg(MHC-MerCreMer) transgenic
mice. Cardiomyocyte-specific Serca2 deletion may be induced by injection of
tamoxifen in adult mice. Tamoxifen-injected Serca2flox/floxTg(MHC-MerCreMer)
and control Serca2flox/flox mice are termed SERCA2KO and SERCA2FF mice,
respectively.
At 4 weeks after tamoxifen induction, the Serca2 gene was efficiently deleted in
SERCA2KO myocardium (<3% Serca2 mRNA and <5% SERCA2 protein).
Functional thapsigargin-sensitive Ca2+ ATPase was reduced to 15 % of SERCA2FF
controls, as measured by 32P-incorporation in tissue homogenates. In isolated
cardiomyocytes, we did not detect SERCA2, SERCA1 or SERCA3 proteins.
Strikingly, SERCA2KO mice did not present clinical signs of circulatory failure at
this timepoint. Contractile parameters were modestly affected, and cardiac output
was 80 % of SERCA2 FF control mice. The left atrium was slightly dilated. We
found no evidence of hypertrophy, disarray, necrosis or fibrosis in histological
sections, nor induction of hypertrophy markers ANP or BNP. However, the lung
weight was slightly increased, suggesting some myocardial dysfunction. Protein
expression of the L-Ca2+ channel alpha2 regulatory subunit, sodium/calcium
exchanger NCX1 and plasma membrane Ca2+-ATPase (PMCA) were all increased
in SERCA2KO mice, suggesting alterations in Ca2+ fluxes over the plasma
membrane. Cardiomyocyte function in 4 week SERCA2KO mice is presented in
the accompanying poster.
In conclusion, the SERCA2KO mouse represents a new and unique phenotype of
Ca2+-handling in vivo. The model should be useful for mechanistic studies of Ca2+
transport, compensatory mechanisms and progressive heart failure in vivo.
27
P17
Increased transsarcolemmal Ca2+ flux in mice with
cardiomyocyte-specific deletion of the Serca2 gene
William E. Louch1,2, Jon Arne Kro Birkeland1,2, Alexandra Vanessa Finsen1,2, Kristin
Brevik Andersson1,2, Ivar Sjaastad1,2,3, Yibin Wang4, Ju Chen5, Jeffery D.
Molkentin6, Kenneth R. Chien7, Ole M. Sejersted1,2, Geir Christensen1,2
1
2
3
4
5
6
7
Institute for Experimental Medical Research, Ullevaal University Hospital, Oslo,
Norway.
Center for Heart Failure Research, University of Oslo, Oslo, Norway.
Department of Cardiology, Heart and Lung Center, Ullevaal University Hospital,
Oslo, Norway.
Departments of Anesthesiology, Physiology and Medicine, David Geffen School
of Medicine, University of California Los Angeles, Los Angeles, CA, USA.
Institute of Molecular Medicine, University of California San Diego, La Jolla,
California, USA.
Department of Pediatrics, University of Cincinnati, Children's Hospital Medical
Center, Division of Molecular Cardiovascular Biology, Cincinnati, Ohio, USA.
Cardiovascular Research Center, Massachusetts General Hospital, Charlestown,
Massachusetts, USA.
Heart failure is associated with reduced activity of the sarcoplasmic reticulum
(SR) Ca2+ ATPase (SERCA) in cardiomyocytes. To investigate the cardiac
phenotype following SERCA downregulation, we recently created a genetically
modified mouse which allows conditional cardiomyocyte-specific excision of the
Serca2 gene. Four weeks after gene deletion SERCA2 knockout mice (SERCA KO)
exhibit very low levels of SERCA2 protein (< 5% control) although the animals do
not exhibit signs of heart failure. Here we examined whether endogenous
mechanisms compensate for reduction in SERCA expression in isolated left
ventricular cardiomyocytes. SERCA KO myocytes exhibited markedly reduced
contraction magnitudes relative to flox-flox controls (SERCA FF) during 1 Hz field
stimulation (SERCA KO = 27% SERCA FF values, P<0.05). Relaxation was also
much slower in SERCA KO cells (maximal relaxation rate = 7% of SERCA FF).
Parallel alterations were observed in Ca2+ homeostasis; Ca2+ transients (fluo-4
AM) were markedly smaller in SERCA KO than SERCA FF and the rate of Ca2+
decline was reduced. The releasable SR Ca2+ content was decreased in SERCA KO
to 27% of SERCA FF values. Comparison of transients in the absence and
presence of caffeine indicated that while SR Ca2+ release contributed to 91% of
the Ca2+ transient in SERCA FF, only 14% of the Ca2+ transient in SERCA KO was
SR-derived. The peak L-type Ca2+ current was 53% larger in SERCA KO than
SERCA FF, and the outward Na+-Ca2+ exchange current assessed during voltage
ramps was increased by between 60% (100 mV) and 103% (60 mV). Thus,
increased cycling of Ca2+ across the sarcolemma in SERCA KO cells compensates
for diminished contribution of the SR to the Ca2+ transient.
28
P18
Role of Serca2 in the embryonic heart
Cecilie Sjåland1,2, Geir Christensen1,2, Kristin Brevik Andersson1,2
1
2
Institute for Experimental Medical Research, Ullevaal University Hospital, Oslo,
Norway.
Center for Heart Failure Research, University of Oslo, Oslo, Norway.
The SR Ca2+-ATPase, SERCA2 plays a major role in transporting Ca2+ from the
cytosol into the SR during the relaxation state of the excitation-contraction cycle
in the heart. Several genetically modified mice have been used to study Serca2
functions in vivo, however homozygous Serca2null die in utero, thus precluding
further analysis of Serca2 function.
To study the Serca2 function specifically in the heart, we have created the
Serca2flox mouse, a genetically modified mouse carrying loxP sites in the Serca2
gene locus Serca2flox such that all Serca2 isoforms would be deleted upon excision
mediated by Cre recombinase. Serca2flox mice were mated with MHC-Cre mice
in order to generate animals with a heart-specific inactivation of the Serca2 gene
from embryonic day 8.
Initial breeding results showed a skewed distribution of inheritance of the
Serca2flox allele and the MHC-Cre transgene. We found that homozygous
knockout Serca2flox/flox Tg(MHC-Cre) embryos died before embryonic day 11.5.
This phenotype was unexpected since studies have shown that the Ca2+
transients in the embryonic cardiomyocyte are generate mainly by the L-Ca2+
channel and Na+/ Ca2+-exchanger (NCX1). Furthermore, that the SR is not fully
developed until late in embryonic life.
We are currently investigating the underlying mechanisms for the unexpected
early death of cardiac Serca2 knockout embryos.
29
P19
Interplay between the systolic Ca transient, SR Ca content
and Ito: a recipe for arrhythmias
K.M. Dibb, H.K. Graham, L.C. Diffley, J.D. Clarke, A.W. Trafford
Unit of Cardiac Physiology, The University of Manchester, UK
Previously we have shown that the reduction in Ca transient amplitude observed
in the hypertrophied heart was brought about by a decrease in SR Ca content. In
this study we have investigated if Ca homeostatic mechanisms are altered in a
similar way during congestive heart failure (CHF) and how arrhythmogenic risk
may be increased despite any reduction in SR Ca content.
Heart failure was induced in adult male ferrets by chronic pressure overload of
the left ventricle. At the onset of CHF (or the equivalent time period for sham
operated animals) myocytes were isolated from the sub-epicardial (epi) and subendocardial (endo) regions of the left ventricle. Experiments were performed
using the perforated/whole cell patch technique under current or voltage clamp
control at 37°C.
Ca transient amplitude was significantly reduced during CHF (680±78 vs
362±51nM sham vs CHF; p<0.05, n=16 to 20). CHF resulted in a reduction in SR
Ca content to 76% of sham levels (85±8.6 vs 65±6.2 µM CHF vs sham; p<0.05,
n=10 to 23) and we suggest this underpins the reduction in Ca transient
amplitude. RT interval (analogous to QT interval) derived from ECG telemetry
implants, was found to increase on progression to CHF and ventricular
arrhythmias were observed. Action potential duration (90% recovery from peak
at 0.5Hz) was increased during CHF in the endo region only (299±25 vs
618±78ms sham vs CHF). The transient outward current (Ito) was decreased in
the endo region during CHF (3.0±0.6 vs 1.9±0.5pA/pF sham vs CHF p<0.05).
This difference did not exist in the epi region and we suggest that regional
alterations in Ito may contribute to the regional changes in action potential
duration and subsequently the increased RT interval at CHF. Enhanced transmural
gradients in action potential duration and Ito may increase the risk of arrhythmias
via re-enterant mechanisms.
30
P20
Reduced t-tubule density and less synchronous Ca2+
release in cardiomyocytes from rats with congestive heart failure
Fredrik Swift1, William E. Louch1, Jon Arne Kro Birkeland1, Marion J. Thomas1,
Mark B. Cannell2, Ivar Sjaastad1, 3, Ole M. Sejersted1
1
2
3
Institute for Experimental Medical Research and Center for Heart Failure
Research, University of Oslo, Ullevaal University Hospital, Oslo, Norway
Department of Physiology, Faculty of Medicine and Health Sciences, University
of Auckland, New Zealand
Department of Cardiology, Heart and Lung Center, Ullevaal University Hospital,
Oslo, Norway
Release of Ca2+ from the sarcoplasmic reticulum (SR) in cardiomyocytes occurs at
functional units where Ca2+ channels located in the t-tubules face SR Ca2+ release
channels. The close proximity of these proteins ensure efficient excitationcontraction coupling. Experimentally reducing t-tubule density has been shown to
increase the dyssynchrony of Ca2+ release. We investigated whether alterations in
t-tubule density and Ca2+ release synchrony occur in cardiomyocytes from rats
with post infarction congestive heart failure (CHF). Post infarction CHF was
induced by ligation of the left coronary artery. Hearts were fixed with a solution
containing 4% paraformaldehyde and mounted in Lowicryl HM20 resin. Thin
sections were cut, mounted onto nickel grids, and randomized for blinded
examination by electron microscopy. Mean density of the t-tubules was lower in
micrographs from failing cardiomyocytes compared to sham (212 t-tubules/100
µm2 vs. 362 t-tubules/100 µm2, p<0.01). T-tubule density was also reduced in
isolated cardiomyocytes labeled with Alexa 488 conjugated wheat germ agglutine
(WGA). Line-scan images of Ca2+ transients (fluo-4 AM, 1Hz) in field stimulated
isolated cardiomyocytes showed that SR Ca2+ release was significantly less
synchronous in failing than in sham cells. The degree of dyssynchrony correlated
with the severity of CHF. Thus, loss of t-tubules during CHF development may
promote dyssynchrony of Ca2+ release, and contribute to decreased efficiency of
excitation-contraction coupling in this disease.
31
P21
Myocardial infarction in adult mice induces hypertrophy
and congestive heart failure without reduced contraction of noninfarcted cardiomyocytes.
Halvor K. Mørk1,2, Ivar Sjaastad1,2,3, Ole M. Sejersted1,2, William E. Louch1
1
2
3
Institute for Experimental Medical Research, Ullevaal University Hospital, Oslo,
Norway
Center for Heart Failure Research, University of Oslo, Oslo, Norway
Department of Cardiology, Heart and Lung Center, Ullevaal University Hospital,
Oslo, Norway
End-stage heart failure in humans is associated with reduced contraction of
individual cardiomyocytes, smaller Ca transients, and reduced Ca content of the
sarcoplasmic reticulum. We investigated whether these alterations were present
in mice with congestive heart failure (CHF). Myocardial infarction (MI) was
induced by ligation of the left coronary artery, and CHF mice were examined at
10-weeks post-MI. SHAM-operated mice served as controls. Heart weight / body
weight ratios were 163±20% higher in CHF than SHAM indicating hypertrophy.
Lung weight / body weight ratios were also increased (CHF 221±30% of SHAM
values) indicating congestion. Echocardiographic analysis showed marked
reductions in cardiac output (SHAM 39±4 ml/min; CHF 25±3 ml/min, P<0.05)
and left-ventricular fractional shortening (SHAM 27±4%; CHF 8±1%, P<0.05).
However, thickening of the non-infarcted posterior wall was unaltered (SHAM
27±6%, CHF 36±7%). Cardiomyocytes isolated from non-infarcted septum were
significantly longer in CHF than SHAM (178±8 µm vs 139±6 µm, P<0.05). During
field stimulation (1 Hz, 37C), myocyte contraction magnitude was similar in CHF
and SHAM cells (% shortening = 9.1±1.1 vs 9.7±0.4, P=NS). Surprisingly, Ca
transients (fluo-4 AM) were larger in CHF than in SHAM at both 1Hz (CHF
F/F0=3.0±0.2, SHAM F/F0=2.3±0.1, P<0.05) and 5 Hz stimulation frequencies,
and the rate of Ca decline was unchanged. Caffeine-elicited transients were of
similar magnitude in CHF and SHAM (F/F0=4.3±0.5 vs 5.0±0.4, P=NS),
suggesting that sarcoplasmic reticulum Ca content was unaltered. Therefore, CHF
progression in mice does not affect contractions of the viable myocardium to the
same extent as in heart failure patients.
32
P22
Differential regulation of genes encoding contractile
proteins during development of heart failure after myocardial
infarction in mice
Cathrine Husberg1,2, Alexandra Finsen1,2, Ståle Nygård1,2,3, Geir Christensen1,2
1
2
3
Institute for Experimental Medical Research, Ullevål University Hospital,
University of Oslo
Center for Heart Failure Research, University of Oslo
Department of Mathematics, University of Oslo
After myocardial infarction (MI), the non-infarcted region of the left ventricle
responds by changing the expression profile of contractile proteins. While the
switch from -myosin heavy chain (MHC) to -MHC expression has been well
described, the regulation of other proteins in the contractile apparatus has been
less studied. We have performed a whole genome profiling of non-infarcted left
ventricular tissue isolated from mice at four different time points after left
coronary artery ligation. The results from the microarray experiments were
evaluated by significance analysis of microarrays (SAM) and the ontological
mapping software High-Throughput GoMiner. Significant regulation of several
genes encoding proteins involved in muscle development and contraction where
identified. Interestingly, the genes encoding signaling molecules (e.g. RhoA,
Cdc42, Smpx) showed an early response, suggesting a more rapid reaction to the
injury than genes encoding contractile proteins. Among the genes coding for
contractile proteins, the most pronounced change was the increased expression
of -MHC (16 fold on day 7 post MI). Regarding the various myosin light chain
(MLC) encoding genes, a diverse regulation pattern was observed with the
highest degree of regulation among regulatory isoforms. Other genes identified
as up-regulated where tropomyosin 4 and skeletal muscle -actin, while troponin
I was constantly down-regulated. The latter is known to affecting muscle
contraction and Ca2+-responsiveness. The results show that whole genome
profiling of the non-infarcted left ventricle enables a better understanding of the
temporal alterations in the expression of the entire contractile machinery during
development of HF. Especially interesting is the extensive alterations in
expression of myosin light chains, which may significantly alter the Ca2+responsiveness of the contractile apparatus.
33
P23
Calcium handling in cardiomyoctes isolated from rats with
post infarction heart failure – effect of hypoxia/reoxygenation
and ouabain exposure.
Mohammad Nouri Sharikabad1, Janne Pedersen1, Ivar Sjaastad2, Ole M.
Sejersted2, Odd Brørs1
1
2
Clinical Chemistry Department, Ullevål University Hospital, Oslo.
Institute for Experimental Medical Research, Ullevål University Hospital, Oslo.
Introduction: Knowledge about calcium handling of failing myocardium during
hypoxia (low oxygen tension) and reoxygenation is important for understanding
the pathology of heart failure and for finding strategies to improve function and
survival of the failing myocardium.
Aim: The purpose of the present study was to investigate calcium accumulation
during reoxygenation of hypoxic cardiomyocytes isolated from rats with postmyocardial-infarction (MI) heart failure compared to control animals. Since influxmode-Na+/Ca2+ exchange is a known candidate for reoxygenation-mediated
calcium uptake, the calcium accumulation was also studied in the presence of
1mM ouabain (an inhibitor of Na+/K+ ATPase causing Na+-loading) in normoxic
conditions.
Methods: Cardiomyocytes were isolated by trypsin/collagenase perfusion of adult
Wistar rat hearts from sham-operated animals and animals with post MI heart
failure. Hypoxia was induced by changing the atmosphere to 95% nitrogen - 5%
CO2 for 4 hours. Cells were then reoxygenated (2 hours) by changing the
atmosphere to 95% air - 5% CO(2) and adding glucose (5.5 mM).
Cardiomyocytes from failing and sham hearts were incubated in normoxic
conditions with the physiological buffer supplied with 1 mM ouabain to study
calcium accumulation during Na+-loading in separate set of experiments. Cell
calcium was measured by 45Ca2+ uptake and was related to protein content.
Results: Hypoxia-reoxygenation caused 6 fold increase in Ca2+-accumulation in
sham cardiomyocytes whereas failing cardiomyocytes did not show any increase.
In the presence of 1mM ouabain (normoxic conditions) both sham and failing
cardiomyocytes show a similar large increase in calcium accumulation (50-70 fold
compared to non-treated cells).
Discussion/conclusion: Our results demonstrate a large difference between
calcium handling of cardiomyocytes from post MI-failing hearts compared with
cardiomyocytes from sham control hearts during hypoxia-reoxygenation.
Reoxygenation-mediated calcium uptake is associated with cell death/dysfunction
in various cellular and intact heart models of reoxygenation/reperfusion.
Interestingly both cell types took up a similar significant amount of calcium in the
presence of ouabain (Na+-loading). In conclusion: The present data indicate
improved tolerance of the failing cardiomyocytes to hypoxia and reoxygenation.
34
P24
Increased amounts of PP1 and PP2A mediating reduced
serine 16 phosphorylation of phospholamban is a possible
mechanism for cardiac dysfunction in alveolar hypoxia
Karl-Otto Larsen1,2,3, Birgitte Lygren4, Ivar Sjaastad1,3,5, Kurt A. Krobert6, Geir
Florholmen1,3, Kristin Arnkværn1,3, Ole Henning Skjønsberg2, Kjetil Taskén4, Geir
Christensen1,3
1
2
3
4
5
6
Institute for Experimental Medical Research, Ulleval University Hospital
Department of Pulmonary Medicine, Ulleval University Hospital
Center for Heart Failure Research, University of Oslo
The Biotechnology Centre, University of Oslo
Department of Cardiology, Ulleval University Hospital
Department of Pharmacology, University of Oslo
Introduction: We have previously reported right ventricular (RV) dysfunction and
left ventricular (LV) diastolic dysfunction in mice subjected to alveolar hypoxia,
and that reduced Serine (Ser) 16 phosphorylated phospholamban (PLB) might be
a mechanism for the RV and LV dysfunction. The mechanisms leading to
hypophosphorylation of PLB in alveolar hypoxia are not known.
Hypothesis: Chronic alveolar hypoxia induces circulating mediators, including
cytokines, which lead to changes in the beta-adrenergic pathway or in
phosphatases causing reduced Ser16 phosphorylated PLB in both ventricles.
Methods: Mice were exposed to 10% oxygen for 2 weeks. Signaling molecules
were measured in cardiac tissue, isolated sarcoplasmic reticulum (SR) and serum.
Results: Ser16 phosphorylated PLB in the RV and LV free wall was reduced in the
hypoxia group to 84 % and 59 % of controls. The levels of beta-adrenergic
receptors and adenylyl cyclase activity were unaltered. The amounts of PKA
regulatory and catalytic subunits (RI, RII and C), and A kinase anchoring
proteins detected by RII-overlay were not changed. However, alveolar hypoxia
induced an increase in the amount of PP1 in the RV and the LV free wall to 143%
and 177 % of controls. In the LV free wall, PP2A was increased to 124 % of
control. The concentrations of IL-18, IL-12p40, RANTES, IL-5, keratinocyte
chemoattractant and GM-CSF were increased in serum after 1 day of hypoxia,
and IL-18 levels were increased after 2 days, 1 and 2 weeks. Hypoxia increased
the amount of Akt, which is involved in IL-18 signaling, to 116 % of control in the
RV.
Conclusions: Given the unchanged -adrenergic pathway, increased amounts of
SR-associated PP1 and PP2A during alveolar hypoxia may be the cause of
reduced Ser16 phosphorylated PLB, depressed SR function and cardiac
dysfunction. It remains to be determined whether increased IL-18 can influence
the observed alterations in phosphatases.
35
Moderated poster session IV: Cardiac and skeletal muscle
dysfunction in heart failure
P25
Left ventricular systolic torsion by 2-D speckle tracking
imaging is load dependent
Anders Hodt, Marie Stugaard, Jonny Hisdal, Einar Stranden, Dan Atar, Kjetil
Steine
P26
Combined treatment with exercise training and losartan
ameliorates metabolic dysfunction in the failing heart
Morten André Høydal, Ole Johan Kemi, Per Magnus Haram, Anne Garnier, Renee
Ventura- Clapier, Øyvind Ellingsen
P27
Fatigue in slow twitch skeletal muscle following a
dynamic exercise protocol
Morten Munkvik, Per Kristian Lunde, Ole M Sejersted
P28
Use of single skinned muscle fibres to study the
mechanism of increased skeletal muscle fatigability in heart
failure
Esther Verburg, Morten Munkvik, Per-Kristian Lunde, Graham D. Lamb, Ole M.
Sejersted
P29
The effect of unilateral knee extensor training on cytokine
expression in skeletal muscle of heart failure patients and
controls
Tommy Aune Rehn, Morten Munkvik, Per Kristian Lunde, Hilde Dishington,
Gunnar Slettaløkken, Bjarne Rud, Martin Søkjer, Jostein Hallen, Ole M. Sejersted
P30
Calcium handling in skeletal muscle from trained and
untrained thigh from heart failure patients and controls
Per Kristian Lunde, Tommy Aune Rehn, Morten Munkvik, Almira Karahasan,
Gunnar Slettaløkken, Jostein Hallén, Ole M. Sejersted
36
P25
Left ventricular systolic torsion by 2-D speckle tracking
imaging is load dependent
Anders Hodt1, Marie Stugaard1, Jonny Hisdal2, Einar Stranden2, Dan Atar1, Kjetil
Steine1
1
2
Divsion of Cardiology, Aker University Hospital, Oslo
Division of Cardiovascular Physiology, Aker University Hospital, Oslo
Background: 2-D ultrasound speckle tracking imaging (STI) is a promising tool to
assess LV rotation and torsion non-invasively, and correlates well with MRI
tagging. The effect of preload on LV torsion is controversial. To explore this issue,
we studied 12 healthy men by lower body negative pressure (LBNP).
Methods: LBNP was applied using a chamber enclosing lower body to reduce
central venous pressure and preload. At baseline and LBNP at -20 and -40
mmHg, LV basal and apical short axes images were acquired to measure LV
rotation by STI. LV torsion was defined as difference of LV rotation between apex
and base. LBNP at -40 mmHg corresponds to reduction in capillary wedge
pressure of about 4 mmHg. Stroke volume (SV) was measured by pulsed Doppler
in the LV outflow tract. LV areas were calculated from endocardial traces of short
axis images.
Results: See table.
Stroke volume, ml
Heart rate, bpm
LV-area end-diastole, cm2
LV-area end-systole, cm2
Peak systolic rotation base, deg
Peak systolic rotation apex, deg
Peak systolic torsion, deg
(Statistical analyses were performed
Baseline
LBNP -20mmHg
77±20
62±11
52±7
52±7
22.3±
20±3
12±2
11±2
-5.5±3.8
-4.5±3.6
4.8±2.3
4.7±1.7
10.5±3.2
9.8±3.0
by repeated measures ANOVA)
LBNP -40mmHg
46±6
57±8
17±3
10±1
-5.7±3.1
3.4±2.4
8.5±3.2
p-value
<0.001
<0.01
<0.001
<0.001
0,486
0.086
0.039
Multiple regression showed that the only independent predictor of peak systolic
torsion was SV, r = 0.59, r² = 0.35, p < 0.05. The figure shows representative
area/torsion loops at baseline (solid line) and LBNP at -40 mmHg (broken line).
Conclusion: Peak systolic torsion was significantly reduced, corresponding to a
decrease in SV. Reduction of torsion seems to be caused by a decrease in apical
rotation. Reduction in preload results in left and downward shift of the
area/torsion loop, which indicates that loading is important for LV torsion.
37
P26
Combined Treatment with Exercise Training and Losartan
Ameliorates Metabolic Dysfunction in the Failing Heart
Morten André Høydal1, Ole Johan Kemi3, Per Magnus Haram1, Anne Garnier4,
Renee Ventura- Clapier4, Øyvind Ellingsen1,2
1
2
3
4
Department of Circulation and Medical Imaging, Faculty of Medicine, Norwegian
University of Science and Technology, Trondheim, Norway
Department of Cardiology, St Olavs Hospital, Trondheim, Norway
Institute of Biomedical and Life, University of Glasgow, UK.
Université Paris Sud, Faculté de Pharmacie, Paris, France
Aim: Current clinical approaches for post-myocardial infarction heart failure
include exercise training and pharmacological strategies. However, few studies
have investigated how combined treatment with those two separates from
exercise training or medication alone.
Method and results: We established cardiac metabolic dysfunction in postmyocardial infarction heart failure, before starting combined or separate
treatment interventions with exercise training and losartan, and Angiotensin II
type I (AT1) receptor antagonist. Following left coronary artery ligation in
Sprague-Dawley rats, losartan was distributed after 1 week, whereas aerobic
interval exercise training at 85-90% of maximal oxygen uptake was initiated 4
weeks post-infarction. Overall, combined treatment yielded best outcome for
ventricular pressure characteristics and exercise capacity. Metabolic dysfunction
was indicated in myocardial tissue in heart failure, as observed by reduced
enzyme activities of creatine and adenylate kinase systems, citrate synthase,
cytochrome c oxidase, and, to a lesser degree, lactate dehydrogenase. Gene
expression levels of cytochrome c oxidase and peroxisome proliferator-activated
receptor gamma coactivator 1  (PGC1), a key regulator of mitochondrial
biogenesis and homeostasis tended to decrease in heart failure. Combinatorial
treatment with exercise training and losartan partly or fully restored metabolic
enzymes and PGC1 gene expression towards healthy levels, whereas exercise
training or losartan alone had less order of magnitude effects; in fact, losartan
alone appeared to further reduce activity in enzymes involved in energy shuttling
and ATP production.
Conclusion: Cardiac metabolic dysfunction in heart failure is ameliorated when
losartan is given jointly with a controlled, rigorous exercise training program.
38
P27
Fatigue in slow twitch skeletal muscle following a
dynamic exercise protocol
Morten Munkvik, Per Kristian Lunde, Ole M Sejersted
Institute for Experimental Medical Research and Center for Heart Failure
Research, University of Oslo, Ullevål University Hospital, N-0407 Oslo, Norway
Fatigue mechanisms seem to be different depending on whether the muscle is
allowed to shorten (isotonic contractions) or not (isometric contractions) during
stimulation. Slow twitch soleus muscles from 12 weeks old male Wistar rats
(370±4 g) were stimulated intermittently at 30 Hz (1 sec on 1 sec off for 15 min;
1 ms pulses) in situ at 37ºC and allowed to shorten after the force had reached
1/3 of maximum force (234±4 g). Resting baseline-tension showed a transient
rise, reflecting a significant slowing of relaxation that peaked after 100 sec, and
returned to near preload values after 5 min. During the first 5 min the shortening
declined gradually to about 30 % of the start values. The last ten minutes
elapsed with no changes in the recorded parameters. The maximal isometric
force-production was reduced after 100 sec, but had regained pre-protocol level
after 15 min. Both exercising (EX) and contralateral resting (REST) muscles were
frozen and analyzed for metabolites after 100 sec and after 15 min of stimulation.
CrP and ATP were significantly lower in EX after 100 sec, but there were no
differences compared to REST after 15 min. A “slack test” showed that unloaded
shortening velocities were lowest after 100 sec (155 mm/s), and approached
pretest values (180 mm/s) after 15 min (165 mm/s), indicating transient changes
in the contractile apparatus. Multiplexed proteomics showed that phosphorylation
of myosin light chain 2 (MLC2) was reduced at 100sec and remained low. We
conclude that isometric muscle dynamics and velocity of unloaded shortening
seem to follow CrP and ATP levels, whereas the isotonic fatigue, seen as reduced
isotonic shortening, follows the MLC2 phosphorylation level.
39
P28
Use of single skinned muscle fibres to study the
mechanism of increased skeletal muscle fatigability in heart
failure
Esther Verburg1, Morten Munkvik1, Per-Kristian Lunde1, Graham D. Lamb2, Ole M.
Sejersted1
1
2
Institute for Experimental Medical Research, Ullevaal University Hospital, Oslo,
Norway
Dept. of Zoology, La Trobe University, Bundoora Campus, Melbourne, Australia
The skinned muscle fibre technique has been used for many years to study the
peripheral mechanism of fatigue in healthy skeletal muscle. The technique has
contributed to clarifying the roles of for example t-tubuli excitability and lactic
acid, ATP, ADP and inorganic phosphate and elevated levels of cytosolic calcium
for the development of fatigue.
The skinned fibre technique involves the dissection of a section of a single muscle
fibre from a whole muscle or a muscle biopsy, followed by the mechanical
removal of the outer membrane (sarcolemma) of the muscle cell. In this process
the t-tubuli and its connections to the sarcoplasmic reticulum remain intact. The
t-tubuli seal off at the sarcolemmal end. The excitation-contraction (EC-) coupling
system is fully functional, and the fibers can be stimulated to contract by either
depolarisation of the t-tubuli or direct activation of the contractile proteins with
Ca2+. Removing the sarcolemma allows free access to the cytosol, and thus the
constitution of the cytosol can be manipulated to investigate the roles of acute
changes in the concentrations of its components. Altered concentrations of
certain ions (for example Mg2+, Na+, K+, Ca2+) or addition of certain components
(for example caffeine), can be used to directly stimulate and test certain steps of
the EC-coupling system. Tests that can be done include amongst others: [Ca2+]force relationship of the contractile proteins, the capacity of the SR to release and
sequester Ca2+, SR Ca2+ content (both total and releasable), force-frequency
relationship (electrical stimulation of the t-tubuli), sensitivity of the EC-coupling
system to altered [ATP], [ADP], [Pi] and [lactic acid], Ca2+-activated protease
(calpain) activity.
In our project at the Institute for Experimental Medical Research, we aim to
investigate whether the different steps in the EC-coupling system and the
mechanism of fatigue in skeletal muscle are altered in heart failure.
40
P29
The effect of unilateral knee extensor training on cytokine
expression in skeletal muscle of heart failure patients and
controls
Tommy Aune Rehn1,2, Morten Munkvik1,2, Per Kristian Lunde1,2, Hilde
Dishington1,2, Gunnar Slettaløkken3, Bjarne Rud3, Martin Søkjer4, Jostein Hallen3,
Ole M. Sejersted1,2
1
2
3
4
Institute of experimental medical research, Ullevål University Hospital
Center for heart failure research
Norwegian school of sport sciences
Radiological department, Ullevål University Hospital
Patients with congestic heart failure (CHF) have increased plasma levels of
proinflammatory cytokines that correlate to disease severity and prognosis.
Several studies have shown that skeletal muscle can produce cytokines. We
hypothesize that skeletal muscle produce inflammatory cytokines to a greater
extent in CHF patients compared to controls and that skeletal muscle is a major
contributor to the inflammatory status in this patient group. In the present study
blood samples and muscle biopsies are taken from CHF patients and controls
before and after 6 weeks of unilateral knee extensor training. During a final
invasive exercise protocol blood samples from the radial artery and both femoral
veins were drawn at rest, during submaximal work of both legs and three
minutes after this work to measure arterio-venous cytokine difference. Arteriovenous difference of IL-1, TNF-, IL-18 and IL-6 over trained and untrained leg
will be compared with immunohistochemistry for localization- and ELISA on
muscle homogenate for quantification of these cytokines. Further, our hypothesis
is that endurance training during which the pumping capacity of the heart does
not limit muscle blood flow, will attenuate the contribution of muscle to increased
circulating levels of proinflammatory cytokines. Preliminary results on IL-18
indicate release from muscle at rest. Interestingly, we see an uptake over
untrained leg and release or reduced uptake over the trained leg during exercise.
Shortly after cessation of exercise release of IL-18 from the untrained leg greatly
exceeded that of the trained leg. It is too early to evaluate possible differences
between groups.
Further, we hypothesize that endurance training were the oxygen delivery to the
working muscle is not limited by the heart’s pumping capacity does not
compromise muscle blood flow will attenuate muscle cytokine production.
Preliminary results indicate a release of IL-18 from muscle at rest. Interestingly,
we see an uptake over untrained leg and a release or lower uptake over trained
leg during exercise. Shortly after ended exercise release of IL-18 from untrained
leg greatly exceeds that from trained leg. It is too early to evaluate possible
differences between groups.
41
P30
Calcium handling in skeletal muscle from trained and
untrained thigh from heart failure patients and controls
Per Kristian Lunde1, Tommy Aune Rehn1, Morten Munkvik1, Almira Karahasan1,
Gunnar Slettaløkken2, Jostein Hallén2, Ole M. Sejersted1
1
2
Institute for Experimental Medical Research, Ullevål University Hospital
Norwegian School of Sport Sciences, Oslo, Norway
We have previously reported reduced fatigue resistance in slow twitch skeletal
muscle from rats with post infarction heart failure (Lunde et al., 2001 and 2002).
However, in contrast to normal muscle intracellular calcium release was not
related to fatigue development in these rats. We therefore hypothesized that
training might affect intracellular calcium cycling differently in muscles from
patients with heart failure as compared with controls. Muscle biopsies were taken
from the thigh muscle vastus lateralis and analyzed both for content of two
isoforms of the calcium pump (SERCA1 and SERCA 2) and the capability of
sarcoplasmic reticulum (SR) to take up and release calcium. The subjects then
trained one leg on an ergometer for six weeks, and another set of biopsies were
taken from vastus lateralis from trained and untrained legs. Endurance of the
trained leg became significantly longer compared to the untrained in both heart
failure patients and controls. As measured by Western blotting technique the
content of the fast calcium pump (SERCA1) was reduced in the trained leg in both
groups as compared to the untrained leg. However, the expression of the slow
calcium pump of the SR (SERCA2) was significantly increased in the trained leg in
both groups.
Conclusion: In both heart failure patients and controls endurance training causes
a reciprocal shift of the expression of the two isoforms of SERCA towards a slower
phenotype. This fits with the prolonged endurance capacity in the trained leg
from the subjects. Thus, in heart failure patients endurance training causes a
normal shift of the reuptake mechanism for calcium into the SR towards slower
rates.
References
Lunde PK, Dahlstedt AJ, Bruton JD, Lännergren J, Thorén P, Sejersted OM,
Westerblad H. Circulation Research 88, 1299-1305. 2001.
Lunde PK, Verburg E, Eriksen M, Sejersted OM. Journal of Physiology 540, 571580. 2002.
42
43
List of participants
Ademaj
Fadil
Univ.Hosp.Centre "Mother TEREZA"
Afzal
Faraz
University of Oslo
Ahmed
M. Shakil
University of Oslo
Andersen
Geir Øystein
Ullevaal University Hospital
Andersson
Kristin B
Ullevaal University Hospital
Andressen
Kjetil Wessel
University of Oslo
Angel
Kristin
Aker University Hospital
Agewall
Stefan
Aker University Hospital
Ariansen
Inger
Ullevaal University Hospital
Arnkværn
Kristin
University of Oslo
Aronsen
Magnus
University of Oslo
Atar
Dan
Aker University Hospital
Attramadal
Håvard
University of Oslo
Aukrust
Pål
Rikshospitalet University Hospital
Austbø
Bjørg
University of Oslo
Bach
Jon
Ullevaal University Hospital
Bjurstrøm
Magne
Ski LegeKontor
Bjørnerheim
Reidar
Ullevaal University Hospital
Bjørnstad
Johannes
Ullevaal University Hospital
Brattelid
Trond
University of Oslo
Brørs
Odd
Ullevaal University Hospital
Buser
Peter
University Hospital Basel, Switzerland
Carlson
Cathrine
Ullevaal University Hospital
Christensen
Geir
Ullevaal University Hospital
Dahl
Mai Britt
Ullevaal University Hospital
Dahl
Christen Peder
Rikshospitalet University Hospital
Damås
Jan Kristian
Rikshospitalet University Hospital
Dibb
Katharine
University of Manchester, UK
Dickstein
Kenneth
University of Bergen
Dishington
Hilde
Ullevaal University Hospital
Doevendans
Pieter
Eikemo
Hilde
University Medical Center, Utrecht, the
Netherlands
University of Oslo
Eisner
David
University of Manchester, UK
Ellingsen
Øyvind
Estensen
Mette-Elise
Norwegian University of Science and
Technology, Trondheim
Rikshospitalet University Hospital
Flatebø
Torun
University of Oslo
44
Florholmen
Geir
Ullevaal University Hospital
Gravning
Jørgen
University of Oslo
Grunnet
Morten
NeuroSearch A/S
Gullestad
Lars
Rikshospitalet University Hospital
Halvorsen
Sigrun
Ullevaal University Hospital
Hansen
Rie Schultz
NeuroSearch A/S
Hasenfuss
Gerd
University of Göttingen, Germany
Haugen
Espen
Ullevaal University Hospital
Hodt
Anders
Aker University Hospital
Hougen
Karina
Ullevaal University Hospital
Husberg
Cathrine
Ullevaal University Hospital
Hussain
Rizwan
University of Oslo
Høydal
Morten
Ihlen
Halfdan
Norwegian University of Science and
Technology, Trondheim
Rikshospitalet University Hospital
Iversen
Per Ole
University of Oslo
Jarstadmarken
Hilde
Ullevaal University Hospital
Karahasan
Almira
Ullevaal University Hospital
Knudsen
Cathrine Wold
Ullevaal University Hospital
Koch
Walter J.
Krobert
Kurt
Jefferson Medical College, Philadelphia,
USA
University of Oslo
Larsen
Karl-Otto
Ullevaal University Hospital
Levy
Finn Olav
University of Oslo
Louch
William E.
Ullevaal University Hospital
Ludewig
Julie
University of Oslo
Lund
Kari
Ullevaal University Hospital
Lunde
Per Kristian
Ullevaal University Hospital
Mann
Douglas
Baylor College of Medicine, Houston, USA
Martinov
Vladimir
University of Oslo
Moltzau
Lise Román
University of Oslo
Munkvik
Morten
University of Oslo
Mørk
Halvor Kjeang
Ullevaal University Hospital
Nilsson
Ullevaal University Hospital
Nouri Sharikabad
Birgitta
Blakstad
Mohammad
Omland
Torbjørn
Akershus University Hospital
Osnes
Jan-Bjørn
University of Oslo
Qvigstad
Eirik
University of Oslo
Rehn
Tommy Aune
Ullevaal University Hospital
Ullevaal University Hospital
45
Riise
Jon
University of Oslo
Rutkovsky
Arkady
Ullevaal University Hospital
Røsjø
Helge
Akershus University Hospital
Sande
Jørn Bodvar
University of Oslo
Sejersted
Ole M.
Ullevaal University Hospital
Sjåland
Cecilie
University of Oslo
Sjaastad
Ivar
Ullevaal University Hospital
Skomedal
Tor
University of Oslo
Solberg
Erik Ekker
Diakonhjemmet Hospital
Solheim
Svein
Ullevaal University Hospital
Stensløkken
Kåre-Olav
Ullevaal University Hospital
Stokke
Mathis
Ullevaal University Hospital
Strømme
Tævje A.
Ullevaal University Hospital
Swift
Fredrik
Ullevaal University Hospital
Toska
Karin
University of Oslo
Trondsen
Roy
Ullevaal University Hospital
Tønnessen
Theis
Ullevaal University Hospital
Valen
Guro
University of Oslo
Vanberg
Paul
Aker University Hospital
Verburg
Esther
Ullevaal University Hospital
Vistnes
Maria
Ullevaal University Hospital
von Lueder
Thomas
Aker University Hospital
Vaage
Jarle
Ullevaal University Hospital
Westheim
Arne
Ullevaal University Hospital
Winer
Lisbeth
Ullevaal University Hospital
Wæhre
Anne
University of Oslo
Yndestad
Arne
University of Oslo
Ødegaard
Annlaug
University of Oslo
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