Original Article

Leukemia inhibitory factor reduces contractile function and inducesalterations in energy metabolism in isolated cardiomyocytes

Geir Florholmen a,*, Vigdis Aas b, Arild Christian Rustan b, Per Kristian Lunde a,Nadine Straumann c, Hilde Eid d, Annlaug Ødegaard a, Hilde Dishington a,

Kristin Brevik Andersson a, Geir Christensen a

a Institute for Experimental Medical Research, Ullevaal University Hospital, University of Oslo, 0407 Oslo, Norwayb Department of Pharmacology, School of Pharmacy, University of Oslo, Oslo, Norwayc Institute for Cell Biology, Swiss Federal Institute of Technology, Zurich, Switzerland

d Center for Clinical Research, Ullevaal University Hospital, University of Oslo, Oslo, Norway

Received 2 July 2004; received in revised form 15 September 2004; accepted 17 September 2004

Abstract

Interleukin (IL)-6 related cytokines may be involved in the pathophysiology of heart failure. Leukemia inhibitory factor (LIF) is anIL-6 related cytokine, and elevated levels of LIF have been found in failing hearts. The aim of our study was to investigate how LIF mayinfluence isolated cardiomyocytes. Adult cardiomyocytes were isolated from male Wistar rat hearts and treated with 1 nM LIF for 48 h.Contractile function was measured using a video-edge detection system. Fractional shortening was reduced at 0.25 Hz in LIF treated cells(7.4% ± 0.5%) compared to control cells (9.0% ± 0.7%). Gene expression analysis showed that expression of the mitochondrial ATP-synthaseF1 a subunit was reduced in cells exposed to LIF. The activity of the enzyme was also reduced in these cells (0.10 ± 0.05 µmol/min per mgprotein) compared to controls (1.23 ± 0.40 µmol/min per mg protein). The levels ofATP and creatine phosphate were reduced by 15.0% ± 3.0%and 11.2% ± 2.7% in LIF treated cells. LIF increased both 3H-deoxyglucose uptake and lactate levels, suggesting an increase in anaerobicenergy metabolism. Beta-oxidation of 14C-oleic acid was increased by 51.2% ± 14.1% following LIF treatment, but no changes were found incellular uptake or oxidation of 14C-oleic acid to CO2. In conclusion, LIF induces contractile dysfunction and changes in energy metabolism inisolated cardiomyocytes.© 2004 Elsevier Ltd. All rights reserved.

Keywords: Isolated cardiomyocytes; Contractile function; Energy metabolism; Leukemia inhibitory factor

1. Introduction

The signals responsible for the pathophysiologicalchanges observed during transition to heart failure are notwell known. Pro-inflammatory cytokines are often activatedin patients with heart failure. Some of these cytokines havebeen shown to induce alterations at the cellular and organlevel, indicating important roles in the pathogenesis of heartfailure [1]. One such cytokine is the interleukin (IL)-6 relatedcytokine leukemia inhibitory factor (LIF). Synthesis of LIF isincreased in failing hearts both at the mRNA and protein

level [2–4]. LIF signals through the gp130/LIF receptor com-plex and activates the Janus kinase (JAK)/signal transducersand activators of transcription (STAT) pathway in cardi-omyocytes [5]. This pathway is involved in hypertrophic[6,7] and cytoprotective [8] responses in cultured neonatalcardiomyocytes.

In a recent study performed on neonatal cardiomyocytes,we found that LIF regulates genes encoding proteins in-volved in energy metabolism [9]. In particular, we observedreduced mRNA expression of a mitochondrial ATP-synthasesubunit. In failing human hearts, reduced mitochondrialfunction has been observed and an imbalance between en-ergy delivery and demand has been suggested [10]. More-over, a reversal to a more fetal energy pattern with increased

* Corresponding author. Tel.: +47-23-01-6800; fax: +47-23-01-6799.E-mail address: geir.florholmen@medisin.uio.no (G. Florholmen).

Journal of Molecular and Cellular Cardiology 37 (2004) 1183–1193

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0022-2828/$ - see front matter © 2004 Elsevier Ltd. All rights reserved.doi:10.1016/j.yjmcc.2004.09.008

reliance on glycolysis has been described in hypertrophiedhearts [11]. Based on our previous gene expression data, wehypothesized that LIF induces functional changes in bothenergy metabolism and contractile function in isolated cardi-omyocytes.

In the present study we therefore investigated effects ofLIF on cell shortening and the enzymatic activity of themitochondrial ATP-synthase, as well as glucose and lipidmetabolism. Our results showed that LIF reduced both con-tractile function and ATP production in isolated adult cardi-omyocytes.

2. Methods

2.1. Cardiomyocyte cultures

Cardiomyocytes from male Wistar rats (250–300 g) wereisolated as described elsewhere [12]. Cells were maintainedin culture for 48 h in medium 199 (M7528, Sigma, St. Louis,MO) with 2 mg/ml bovine serum albumin (BSA), 2 mMDL-carnitine, 5 mM creatine, 5 mM taurine, 0.1 µM insulin,10–10 M triodothyronine (all from Sigma), 100 U/ml penicil-lin (Invitrogen, Paisley, UK) and 100 µg/ml streptomycin(Invitrogen) with or without 1 nM LIF (Chemicon, Te-mecula, CA). Cells were plated on laminin (10 µg/ml; Invit-rogen) in medium 199 (Sigma). Approximately 75% of con-trol and LIF treated cells remained rod-shaped after 48 h inculture in serum-free medium, without the pseudopodia-likestructures associated with dedifferentiation [13,14].

2.2. Assessments of contractile function

Isolated cardiomyocytes were plated on coverslips coatedwith laminin and maintained in culture for 48 h. The equip-ment and experimental setup used for measurements of con-tractile function of the cardiomyocytes have been describedpreviously [12]. In brief, cells were superfused with a Ty-rodes solution (in mM) (5 HEPES, 140 NaCl, 1.8 CaCl2,5.4 KCl, 0.5 MgCl2, 5.5 glucose, 0.4 NaH2PO4, pH 7.4) for2 min before the start of measurements. Cells without blebsor other morphological alterations that responded with con-traction to electric field stimulation were selected for mea-surements of contractile function. Experiments on each cellwere carried out at stimulation frequencies of 0.25, 0.50,1.00 and 2.00 Hz at a temperature of 37.0 ± 0.1 °C. Theexperiments were blinded regarding treatment. Mean con-traction curves were calculated at each frequency from10 consecutive contractions for each cell. Calculations offractional shortening, maximal contraction and relaxationvelocities were performed with Clampfit 9.0 (Axon Instru-ments Inc., Union City, CA). Cell size was measured with asoftware program (Kontron KS100, Carl Zeiss VisionGmbH, Hallbergmoos, Germany).

The acute effect of LIF on fractional shortening was mea-sured using freshly isolated cells electrically field stimulated

at 1 Hz. Cells were first plated onto coverslips coated withlaminin and superfused with Tyrodes solution for 4 min.Then, the cells were superfused for 4 min with Tyrodessolution containing 1 nM LIF (Chemicon). Cell shorteningwas assessed as described above.

2.3. Filter array analysis and Northern blotting

Isolated cardiomyocytes were cultured for 48 h on 94 mmPetri dishes (Greiner Labortechnik GmbH, Frickenhausen,Germany) coated with laminin. Isolation of mRNA, filterarray (Atlas Rat 1.2 Array, Clontech Inc., Palo Alto, CA)preparation and analysis is described elsewhere [9]. A total offive experiments were included in the analysis. Genes withspot intensities within 10% above background were regardedas not expressed. Genes were ranked by ratio for each experi-ment. The binomial probability function was used to calcu-late the probability of obtaining false positives. A P-value of0.026 was calculated if a gene was located above the 85thpercentile or below the 15th percentile in three or moreexperiments. Genes fulfilling these criteria were regarded asdifferentially expressed.

For Northern blotting, isolated cardiomyocytes were cul-tured on 94 mm Petri dishes (Greiner Labortechnik) coatedwith laminin, and followed procedures previously described[9]. Sequenced EST clones and gene-specific primers wereused for the amplification of hybridization probes. Mousemitochondrial ATP-synthase F1 a subunit was PCR amplifiedfrom EST clone IMAGE 3986722. The probe for GAPDHwas provided by H. Prydz, University of Oslo, Norway.

2.4. Assessment of mitochondrial ATP-synthase activity

The enzymatic activity of the mitochondrialATP-synthasecomplex was measured using a spectrophotometric assay asdescribed elsewhere [15,16]. In brief, cells were plated insix-well culture dishes (Corning Int., Corning, NY) coatedwith laminin and maintained in culture for 48 h. Cells werethen washed twice in Buffer 1 (in mM) (25 HEPES,110 NaCl, 2.6 KCl, 1.2 KH2PO4, 1.2 MgSO4, 1.0 CaCl2, pH7.4) and once in Buffer 2 containing (in mM) (20 HEPES,1.0 MgCl2, 2.0 EGTA, pH 7.0). Then, cells were sonicated inBuffer 2 for 3 × 10 s in room temperature with 10 µmamplitude. Samples were kept on ice until measurements.Aliquots were added to a buffer (in mM) (60 sucrose, 50 tri-ethanolamine–HCl, 50 KCl, 4 MgCl2, 2 ATP, 1.5 phospho-enolpyruvate, 2 EGTA, 1 KCN, pH 8.0 with KOH) supple-mented with 200 µM NADH, 5 U pyruvate kinase (P7889,Sigma) and 5 U lactate dehydrogenase (L3888, Sigma). Theconversion of NADH to NAD+ was followed spectrophoto-metrically at 340 nm at 37 °C for 2 min. Oligomycin(10 µg/ml; O4876, Sigma) was used to block the mitochon-drial ATP-synthase. Mitochondrial ATP-synthase activitywas calculated as the difference between total and oligo-mycin-insensitive conversion of NADH to NAD+. NADH

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oxidase activity in the samples was measured using the samebuffer, but without lactate dehydrogenase and pyruvate ki-nase.

2.5. Measurements of ATP, creatine phosphate (CrP)and lactate

Isolated cardiomyocytes were plated on OmniTrays(Nalge Nunc Int., Rochester, NY) coated with laminin. After48 h in culture with or without LIF treatment, the mediumwas removed and cells lysed in 3 M ice-cold perchloric acid(PCA). The ATP and CrP levels were measured by lumines-cence spectrometry and lactate levels measured by fluores-cence spectrometry (model LS50B, Perkin–Elmer, Bucking-hamshire, UK) as previously described [17].

We also measured ATP, CrP and lactate content afterelectric field stimulation. Cells were plated on OmniTrays(Nagle Nunc Int.) and maintained in culture for 48 h with orwithout LIF. Cells were stimulated by graphite electrodeswith bipolar pulses for 45 min at 5 Hz. More than 80% of therod-shaped cells responded with contractions. Cells werelysed in 3 M ice-cold PCA within 10 s after cessation ofstimulation.

2.6. Measurements of cellular content of NADHand NAD+

Measurements of NADH and NAD+ were performed byenzyme coupled measurements of NADH fluorescence asdescribed elsewhere [17]. NADH was excited at 340 nm andemission was measured at 460 nm (fluorometer modelLS50B, Perkin–Elmer). For NADH measurements, cellswere harvested in phosphate buffered saline (PBS) and thenlysed in 0.04 M NaOH. Samples were heated for 10 min at60 °C to remove NAD+ and then stored at –80 °C untilanalysis. The conversion of NADH to NAD+ was followed ina buffer [60 mM Na2HPO4 and 40 mM NaH2PO4 (both fromMerck) at pH 7.0] using pyruvate (1 mM; P2256, Sigma) assubstrate and catalyzed by lactate dehydrogenase(0.5 mg/ml; L2625, Sigma). The decline in fluorescenceintensity was used to calculate NADH content. For NAD+

measurements, cells were harvested in PBS and lysed in abuffer [1.2 M ascorbic acid, 0.02 M H2SO4 and 0.1 MNa2SO4 (all from Merck)]. Samples were heated for 30 minat 60 °C to remove NADH. The conversion of NAD+ toNADH was followed in a buffer (80 mM Tris–HCl, pH 8.7)using ethanol (5%) as substrate and catalyzed by alcoholdehydrogenase (3 mg/ml; A3263, Sigma). The increase influorescence intensity was used to calculate NAD+ content.For NADH and NAD+ measurements, a calibrated NADH(N8129, Sigma) standard was used as internal standard. Mea-surements were performed in triplicates for each sampleusing cells from five experiments.

2.7. Measurements of creatine kinase (CK) activityand CK protein analysis

Cells were plated in six-well culture dishes (Corning Int.)and then lysed in a CK-buffer (1% NP-40, 0.25% deoxycho-lat, 50 mM NaHPO4·H2O, 0.5 mM b-mercaptoethanol) after48 h in culture. For CK activity assay, 64 and 320 ng cellextract in 1 and 5 µl volume, respectively, were added to100 µl assay mix (1.95 ml TRAP, 0.1 M triethanolamine pH7, 1 mM b-mercaptoethanol), 250 µl solution I (2 M glucose,50 mM NADP), 125 µl solution II (200 mM ADP, 500 mMMgCl2), 25 µl enzyme mix (300 U/ml hexokinase (Roche,Basel, Switzerland), 150 U/ml glucose-6-phosphate-dehydrogenase (Roche), 0.4 µl b-mercaptoethanol, 40 µMAp5A) and equilibrated at 30 °C for 10 min. The CK reactionwas started by adding 5 µl 2 M phosphocreatine (Calbio-chem, Switzerland), also pre-equilibrated at 30 °C. The ki-netics of the CK reaction was monitored online by measuringreduction of NADP to NADPH at 340 nm for 30 min at 30 °Cin a Spectra Max 190 (Molecular Device) ELISA-reader.

For Western blot analysis, 20 µg of lysates from controland LIF treated cells were separated on 4–12% SDS gels andtransferred to PVDC membrane overnight in a wet blottingtank. Western blots were probed with antibodies againstbrain-type CK (MaB-CK, kindly provided by B. Wieringa,University of Nijmegen, The Netherlands), ubiquitous mito-chondrial CK (RauMt-CK) [18] and muscle-type CK (RaM-CK) [19] for 1 h at ambient temperature.

2.8. Measurement of nitric oxide (NO) production

NO production was assessed by measuring nitrate andnitrite (NOx) in the culture medium. Isolated cardiomyo-cytes were plated in six-well culture dishes (Corning Int.)coated with laminin. Culture medium was collected after 2 or48 h and centrifuged to remove any cells. Samples werestored at –20 °C until analysis. NOx was analyzed usingTotal Nitric Oxide Assay kit (R&D, Abingdon, UK). Briefly,this assay is based on the enzymatic conversion of nitrate tonitrite by nitrate reductase. To minimize interference withplasma proteins, the samples were ultrafiltrated through a12 kDa cut-off filter (VectaSpin Micro 12K MWCO, What-man International Ltd., Maidstone, UK) prior to the analysisof NOx. The reaction is followed by a colorimetric detection(540 nm) of nitrite as an azo dye product of the Griessreaction. Using a similar method it has earlier been shownthat cells in culture respond with a significant increase in NOproduction after treatment with lipopolysaccharide extracts[20].

2.9. Glucose metabolism

2.9.1. Glucose uptake

Isolated cardiomyocytes were plated on six-well culturedishes (Corning Int.) coated with laminin. Measurements of

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3H-deoxyglucose uptake was performed by a method slightlymodified from Gaster et al. [21]. In brief, the cells weremaintained in culture for 48 h with or without 1 nM LIF, andthen washed twice with uptake buffer (in mM) (140 NaCl,20 HEPES, 5 KCl, 2.5 MgSO4, and 1 CaCl2). Then, the cellswere incubated in the same buffer supplemented with2-[3H(G)]deoxy-D-glucose [104A, American RadiolabeledChemicals (ARC), Inc., MO] (10 µM, 1 µCi/ml). In eachexperiment, a set of control cells was incubated with theuptake buffer also containing 1 µM insulin (Actrapid, Novo-Nordisk, Bagsværd, Denmark). After 15 min, the dishes wereplaced on ice, washed three times with ice-cold PBS, lysedwith 0.05 M NaOH and sonicated. Uptake of labeled glucosewas measured by liquid scintillation. Non-carrier mediateduptake was determined in the presence of 5 µM cytochalasinB (Sigma) and subtracted from all values.

2.9.2. Glucose oxidation

Isolated cardiomyocytes were plated on 12.5 cm2 flasks(Falcon, Becton Dickinson, Franklin Lakes, NJ) coated withlaminin and maintained in culture for 48 h. The control andLIF treated cells were then incubated with D-[1-14C]glucose(120A, ARC; 0.2 µCi/ml, 5.5 mM) and 20 mM HEPES.Flasks were made airtight using stopper tops. After 4 h,300 µl phenyl ethylamine/methanol (1:1, v/v) was added witha syringe to a center well containing a folded filter paper.Three hundred microliters 1 M PCA was subsequently addedto the cells through the stopper tops by use of a syringe. Theflasks were placed for a minimum of 4 h at room temperatureto trap labeled CO2. The filter paper was counted by liquidscintillation. Flasks containing no cells were used to correctfor non-specific CO2-trapping.

2.10. Lipid metabolism

2.10.1. Oleic acid uptake, distribution and oxidation

Isolated cardiomyocytes were plated on six-well culturedishes (Corning Int.) coated with laminin. After 48 h inculture, control and LIF treated cells were incubated with[1-14C]oleic acid (297, ARC) (0.5 µCi/ml, 0.6 mM) for 4 hbefore being harvested in ice-cold PBS, centrifuged (1000 ×g, 5 min), resuspended in distilled water and sonicated. Cellassociated lipids were extracted with chloroform/methanolas described elsewhere [22]. In brief, 400 µl of cell homoge-nate was mixed with 8 ml chloroform/methanol (2:1). FCS(30 µl) was added as a carrier. After 30 min, 1.6 ml 0.9%NaCl was added and the mixture centrifuged (1000 × g,5 min). The organic phase was evaporated under nitrogen at40 °C. The residual lipid extract was redissolved in 200 µlhexane and separated by thin-layer chromatography usinghexane/diethylether/acetic acid (65:35:1) as mobile phase.The bands were visualized with iodine, excised and countedby liquid scintillation.

b-Oxidation of 14C-oleic acid (ARC) was measured asacid soluble metabolites (ASM) (fatty acid b-oxidation prod-

ucts). The labeled 14C-oleic acid isotope forms labeledacetyl-CoA, free acetate, citric acid cycle intermediates andCO2. Control and LIF treated cells were maintained in cul-ture for 48 h on six-well culture dishes (Corning Int.) andthen incubated with 14C-oleic acid for 4 h. ASM were alsomeasured in cells preincubated with 20 µM etomoxir (Re-search Biochemicals International, MA), an inhibitor of car-nitine palmitoyl transferase-1, for 15 min before the additionof 14C-oleic acid. Two hundred and fifty microliters of thecell medium was precipitated with 100 µl 6% BSA and 1.0 ml1 M PCA. After centrifugation (1800 × g, 10 min), 500 µl ofthe supernatant was counted by liquid scintillation. No-cellcontrols were included.

Complete oxidation of oleic acid to CO2 was measured inisolated cardiomyocytes plated on 12.5 cm2 culture flasks(Falcon) coated with laminin. Following 48 h in culture, cellstreated with LIF or controls were incubated with 0.5 mML-carnitine (Sigma), 20 mM HEPES and 14C-oleic acid(ARC) (0.25 µCi/ml, 0.6 mM). Flasks were made airtightusing stopper tops and CO2-trapping followed procedures asdescribed above. Total oleic acid uptake was calculated as thesum of lipids, ASM and oxidation to CO2.

2.11. Statistics

Comparisons of contractile function between control andLIF treated cells at the various stimulation frequencies wereperformed by measuring area under the curve as describedelsewhere [23]. Mann–Whitney rank sum test was used tocompare results from experiments on energy metabolism andNorthern blots. Bonferroni-corrections were performedwhen appropriate. A P-value < 0.05 was considered statisti-cal significant. Data are presented as mean ± S.E.M.

3. Results

3.1. Cardiomyocyte shortening following treatment withLIF for 48 h

Experiments were performed to investigate possible ef-fects of LIF on contractile function in adult cardiomyocytes.We found that 48 h treatment with LIF reduced cardiomyo-cyte fractional shortening when examined in a LIF-free solu-tion at 0.25, 0.50, 1.00 and 2.00 Hz (Fig. 1). At 0.25 Hz, LIFtreated cardiomyocytes had significantly reduced fractionalshortening (7.4% ± 0.5%) versus control cells (9.0% ± 0.7%;P < 0.05). At all frequencies tested, we observed a similarreduction in fractional shortening in LIF treated cells(Table 1). Maximal velocity of shortening was also reducedin cells exposed to LIF (0.21 ± 0.01 µm/ms) versus control(0.26 ± 0.02 µm/ms) at 0.25 Hz (P < 0.05). Similar reductionsin maximal shortening velocities were also observed at theother frequencies tested (Table 1). In LIF treated cardiomyo-cytes, maximal relaxation velocity (0.16 ± 0.22 µm/ms) was

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also reduced compared to control cells (0.22 ± 0.02 µm/ms)at 0.25 Hz (P < 0.05).

We did not detect any significant changes in cardiomyo-cyte resting length during the stimulation protocol. Cell areawas not significantly altered in LIF treated cells (2510 ±73 µm2) following 48 h in culture compared to control cells(2624 ± 58 µm2).

The acute effects of LIF on cardiomyocyte shortening wasinvestigated by adding LIF (1 nM) to the Tyrodes solution. Incontrast to 48 h treatment, we observed no significant changein fractional cell shortening at 1 Hz during superfusion withTyrodes solution containing LIF compared to regular Ty-rodes solution (data not shown).

3.2. Analysis of gene expression following treatment withLIF for 48 h

To identify alterations in gene expression in LIF treatedcells, we used a filter array approach where each filter con-tains cDNA spots representing 1176 different genes. In fiveindependent experiments we found that 264 genes were ex-pressed. Based on our criteria for assessment of differentially

regulated genes, we identified 10 genes with significantlyincreased expression and four genes with significantly re-duced expression (Table 2). Several of the genes with in-creased expression encode receptors and proteins related tocytokine signaling (e.g. tumor necrosis factor receptor 1,transforming growth factor-b II receptor, macrophage colonystimulating factor 1 receptor). One of the downregulatedgenes was the mitochondrial ATP-synthase F1 a subunit.Since downregulation of this gene may influence mitochon-drial energy production, we chose to verify and quantitate thereduced expression of this subunit by Northern blotting. Wefound that the expression in cells treated with LIF was re-duced by 17% ± 1% (P < 0.05) compared to control cells(Fig. 2A).

3.3. Mitochondrial ATP-synthase activity

We further examined if LIF also induced changes in theenzymatic activity of the mitochondrial ATP-synthase. Our

Table 1Cardiomyocyte shortening following 48 h in culture

0.25 Hz 0.50 Hz 1.0 Hz 2.0 Hz OveralleffectCtr LIF Ctr LIF Ctr LIF Ctr LIF

Fractional shortening(%)

9.0 ± 0.7 7.4 ± 0.5 * 6.5 ± 0.5 5.2 ± 0.3 * 4.5 ± 0.3 3.3 ± 0.2 * 3.3 ± 0.3 2.2 ± 0.2 * P < 0.01

Max contractionvelocity (µm/ms)

0.26 ± 0.02 0.21 ± 0.01 * 0.25 ± 0.01 0.20 ± 0.01 * 0.23 ± 0.01 0.18 ± 0.01 * 0.21 ± 0.01 0.16 ± 0.01 * P < 0.01

Max relaxationvelocity (µm/ms)

0.22 ± 0.02 0.16 ± 0.01 * 0.20 ± 0.02 0.15 ± 0.01 * 0.15 ± 0.01 0.10 ± 0.01 * 0.12 ± 0.01 0.08 ± 0.01 * P < 0.01

Cardiomyocytes were electric field stimulated from 0.25 to 2.0 Hz. Mean resting cell length was 100.0 ± 1.0 and 98.1 ± 1.1 µm for control cells (Ctr) and forcells treated with LIF (1 nM), respectively, during measurement of contractile function. Values are mean ± S.E.M. n = 29 and 31 for Ctr and LIF, respectively.* = P < 0.05 for LIF vs. control.

Fig. 1. Effect of LIF (1 nM) on fractional shortening compared to controlcells (Ctr) following 48 h in culture. Each cell was electric field stimulated at0.25–2.00 Hz. n = 31 and 29 for LIF and Ctr, respectively. Values are mean ±S.E.M. * = P < 0.05 vs. Ctr.

Table 2Differentially regulated genes following 48 h in culture

AccessionUpregulatedTumor necrosis factor receptor 1 precursor M63122Protein tyrosine phosphatase, non-receptor type substrate 1 D85183Interferon induced protein X61381Transforming growth factor-b II receptor precursor L09653Neurotensin receptor type 2 X97121Colony stimulating factor 1 receptor X61479ATPase Na+/K+ transporting b 1 polypeptide J02701Cathepsin B X82396Proliferating cell nuclear antigen Y00047PDGF-associated protein U41744DownregulatedMitochondrial ATP-synthase F1 a subunit X56133Muscle phosphofructokinase U25651Guanine nucleotide-binding protein b subunit 3 L29090Cysteine-rich protein 2 D17512

Differentially regulated genes from filter array experiments (n = 5) of car-diomyocytes treated with LIF (1 nM) and control. Expressed genes wereranked by ratio and upregulated genes are located above the 85th percentilein three or more experiments. Downregulated genes are located below the15th percentile in three or more experiments. Accession = GenBank acces-sion number.

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assay showed that the total conversion of NADH to NAD+

was 2.29 ± 0.88 and 0.45 ± 0.09 µmol/min per mg protein forcontrol and LIF treated cells, respectively. The inhibition ofactivity by oligomycin is used as a measure of the mitochon-drial ATP-synthase activity [16]. This approach showed thatthe activity of the mitochondrial ATP-synthase was substan-tially reduced in cells treated with LIF (0.10 ± 0.05 µmol/minper mg protein) compared to control cells (1.23 ±0.40 µmol/min per mg protein; P < 0.05; Fig. 2B). NADHoxidase activity was found to be below 0.2 µmol NADH permin per mg protein.

3.4. ATP and CrP in LIF treated cells before and afterhigh frequency stimulation

Since we observed a reduction in the mitochondrial ATP-synthase activity, we measured the content of ATP and CrP incardiomyocytes following 48 h in culture with or withoutLIF. Our measurements showed a 15.0% ± 3.0% (P < 0.01)reduction of ATP in cells treated with LIF (20.8 ±6.1 nmol/mg protein) versus controls (24.2 ± 6.6 nmol/mgprotein; Fig. 3A). The CrP content was reduced by 11.2% ±2.7% (P < 0.01) in these cells (27.7 ± 10.0 nmol/mg protein)

compared to control cells (32.3 ± 12.3 nmol/mg protein;Fig. 3B).

ATP and CrP levels were also measured after high fre-quency electrical field stimulation of cardiomyocytes follow-ing 48 h in culture. In cells treated with LIF the ATP content(18.2 ± 4.3 nmol/mg protein) was reduced by 16.3% ± 4.4%(P < 0.01) compared to control cells (22.2 ± 5.3 nmol/mgprotein) after electric field stimulation for 45 min at 5 Hz(Fig. 3A). The CrP levels were reduced by 17.3% ± 4.0% (P< 0.01) in the LIF treated cells (25.3 ± 8.5 nmol/mg protein)versus control (30.1 ± 9.5 nmol/mg protein; Fig. 3B).

Fig. 2. (A) Gene expression assessed by Northern blotting. ATP-synthase asubunit = mitochondrial ATP-synthase F1 a subunit. GAPDH = glyceralde-hyde 3-phosphate dehydrogenase. (B) Mitochondrial ATP-synthase activityfollowing 48 h in culture. The reported activities are sensitive to oligomycin(10 µg/ml). n = 5. Ctr = control, LIF = leukemia inhibitory factor (1 nM).Values are mean ± S.E.M. * = P < 0.05.

Fig. 3. (A) Cellular ATP content and (B) cellular CrP content. Values arerelative to control after 48 h in culture for each experiment. EFS = electricfield stimulation for 45 min at 5 Hz. Ctr = control, LIF = leukemia inhibitoryfactor (1 nM). n = 6. Values are mean ± S.E.M. * = P < 0.05.

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3.5. NADH and NAD+ content

Measurements of NADH and NAD+ showed no signifi-cant difference in the NADH/NAD+ ratio between control(78.0 ± 4.0) and LIF treated cells (82.7 ± 18.3). The NAD+

content was not significantly different between control cells(6.9 ± 2.8 nmol/mg protein) and LIF treated cells (6.9 ±0.6 nmol/mg protein). NAD+ measurements were verifiedusing HPLC analysis (data not shown). There was no differ-ence in the NADH content between control and LIF treatedcells (553.9 ± 29.6 and 627.0 ± 61.8 nmol/mg protein forcontrol and LIF treated cells, respectively).

3.6. CK activity and protein content

Alterations in CK activity could be an explanation for theobserved changes in ATP and CrP levels. Our results showed,however, that CK activity was not different between thegroups following 48 h in culture (0.38 ± 0.06 and 0.40 ±0.10 µmol/min per mg protein for control and LIF treatedcells, respectively). No change in the expression of the brain-type, ubiquitous or muscle-type mitochondrial CK isoformswas detected between control and LIF treated cells following48 h in culture (data not shown).

3.7. Formation of NOx in cultured cardiomyocytes

Since the changes in myocyte function and ATP produc-tion could be caused by increased NO production, we mea-sured the formation of NOx in the LIF treated cells. Nosignificant change in NOx formation by LIF (3.0 ±1.2 nmol/mg protein) was found compared to control cells(2.6 ± 1.0 nmol/mg protein) after 2 h in culture or after 48 h inculture (6.9 ± 1.4 and 7.1 ± 2.6 nmol/mg protein for LIFtreated and control cells, respectively).

3.8. Lactate production

The reported changes in ATP production may be partlycompensated by increased anaerobic energy metabolism. Wetherefore measured the lactate content in cardiomyocytes andthe culture medium. The lactate content increased signifi-cantly by 25.5% ± 6.1% (P < 0.01) in LIF treated cells (115.9± 25.7 nmol/mg protein) versus control cells (83.5 ±18.4 nmol/mg protein) following 48 h in culture (Fig. 4A).Lactate secretion to the culture medium increased by 31.7%± 5.6% (P < 0.01) following LIF treatment (103.1 ± 23.2 µM)compared to control (80.0 ± 10.3 µM; Fig. 4B).

3.9. Glucose and oleic acid metabolism

Cardiomyocytes examined after 48 h treatment with LIFhad a 33.3% ± 7.4% (P < 0.01) increase in 3H-deoxyglucose

uptake versus control cells (Fig. 5). The basal 3H-deoxy-glucose uptake in control cells was 5.41 ± 0.48 pmol/mgprotein per min. The increase in 3H-deoxyglucose uptake inLIF treated cells was comparable to the acute effect of 1 µMinsulin which increased the uptake by 43.9% ± 7.0% (P< 0.01) compared to untreated cells. We did not observe asignificant difference in 3H-deoxyglucose uptake betweenthe LIF treated cells and our insulin controls (P = 0.33).Despite elevated uptake of 3H-deoxyglucose following LIFtreatment, there was no significant change in the oxidation of14C-glucose to CO2 (10.2 ± 1.8 nmol/mg protein per h)

Fig. 4. (A) Cellular lactate content and (B) lactate concentration in culturemedium. Values are relative to control after 48 h in culture for each experi-ment. EFS = electric field stimulation for 45 min at 5 Hz. Ctr = control, LIF= leukemia inhibitory factor (1 nM). n = 6. Values are mean ± S.E.M.* = P < 0.05.

1189G. Florholmen et al. / Journal of Molecular and Cellular Cardiology 37 (2004) 1183–1193

compared with control cells (9.3 ± 1.8 nmol/mg proteinper h).

Incubation with LIF also induced changes in lipid metabo-lism. However, we did not observe significant alterations incellular uptake of 14C-oleic acid (Table 3), nor changes in theintracellular distribution of 14C-oleic acid. Instead, we foundthat b-oxidation of 14C-oleic acid, as assessed by ASM, wasincreased by 51.2% ± 14.4% (P < 0.05) in LIF treated cells.We performed additional experiments in order to identify theorigin of the increased b-oxidation in LIF treated cells. Theinhibitor etomoxir blocks mitochondrial carnitine palmitoyltransferase-1 activity. The LIF induced increase inb-oxidation was reduced by 39.8% ± 4.0% in etomoxirtreated cells (20 µM) and reached values not significantlydifferent from control cells. Despite the higher b-oxidationactivity, LIF did not increase the complete oxidation of 14C-oleic acid to CO2 compared to control (Table 3).

4. Discussion

We have in the present study investigated the effects ofLIF on isolated adult rat cardiomyocytes. Treatment with LIFfor 48 h reduced contractile function in these cells. Geneexpression analysis showed that LIF reduced expression ofthe mitochondrial ATP-synthase F1 a subunit. The enzymaticactivity of this enzyme was also reduced, and the contents ofATP and CrP were lower in cells exposed to LIF. CK activityand the NADH/NAD+ ratio were not changed. LIF increasedthe glucose uptake and cellular lactate content, but not glu-cose oxidation. LIF also changed the lipid metabolism byincreasing b-oxidation of oleic acid in the cardiomyocytes.

Following treatment with LIF for 48 h in culture, thecardiomyocytes displayed significantly reduced fractionalshortening accompanied by a reduction in the maximal con-traction velocity at all frequencies tested when compared tocontrol cells. The relaxation velocity of the cardiomyocyteswas also reduced. To the best of our knowledge this is the firststudy that has observed effects of LIF on contractile functionin adult cardiomyocytes. Previous studies have observedother effects of LIF on cardiomyocytes such as growth andanti-apoptosis [6–8].

The contractile function of LIF treated cardiomyocyteswas in the present study examined in LIF-free medium, andthus it is unlikely that reduced shortening was due to acuteeffects of LIF. This notion is supported by our experimentsand by previous findings [3] showing that LIF had no acuteeffect on contractile function. The signaling pathways andmechanisms involved in induction of long-term effects oncell shortening are not known. One recent study suggestedthat activation of the JAK2/STAT3 pathway was involved inIL-6 induced contractile dysfunction [24]. Thus, it is possiblethat the long-term effects of LIF are mediated by STAT3 in-duced transcriptional changes.

We therefore hypothesized that the reduction in myocytefunction after 48 h treatment with LIF is caused by alter-ations in gene expression. Gene expression analysis of LIFtreated cardiomyocytes with cDNA filter arrays showed sig-nificant changes in expression of genes involved in cytokinesignaling and energy metabolism. In a previous study, wefound similar changes in gene expression in neonatal cardi-omyocytes, and that LIF downregulated the expression of theF1 a subunit of the mitochondrial ATP-synthase complex [9].

Our experiments also showed that cells treated with LIFfor 48 h had substantially reduced mitochondrial ATP-synthase activity. Furthermore, these cells had reduced cellu-lar contents of ATP and CrP. This is to our knowledge the firsttime it has been shown that LIF has effects on energy produc-tion in cardiomyocytes. Other cytokines such as IL-1b havealso been shown to reduce ATP levels in isolated cardiomyo-cytes, possibly through NO-mediated inhibition of mito-chondrial enzymes [25]. However, we were not able to detectsignificant differences in NOx production by LIF after 2 or

Fig. 5. Uptake of 3H-deoxyglucose (15 min) and oxidation of 14C-glucose toradiolabled CO2 (4 h). Values are relative to control after 48 h in culture foreach experiment. Ctr = control, LIF = leukemia inhibitory factor (1 nM),insulin = addition of insulin (1 µM) to the buffers used for control cellsfollowing 48 h in culture. Values are mean ± S.E.M. * = P < 0.05.

Table 3Metabolism of oleic acid in cardiomyocytes

Ctr LIFOleic acid uptake 149.4 ± 12.4 160.6 ± 13.6Free oleic acid 23.3 ± 3.8 25.6 ± 4.7Diacylglycerol 6.5 ± 0.5 6.8 ± 0.6TAG 19.0 ± 2.1 17.9 ± 1.6Phospholipid 14.2 ± 0.8 15.5 ± 0.6ASM 25.9 ± 8.3 35.7 ± 8.5 *Oxidation to CO2 60.0 ± 13.0 58.7 ± 13.8

Metabolism of [1-14C]oleic acid (0.5 µCi/ml, 0.6 mM) for 4 h following48 h in culture with or without LIF (1 nM). Oleic acid uptake was measuredas the sum of lipids, ASM and oxidation to CO2. Beta-oxidation of oleic acidwas measured as ASM. Values are given as nmol/mg protein (mean ±S.E.M.). Ctr = control, LIF = leukemia inhibitory factor. * = P < 0.05, n = 5.

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48 h in culture. Our results therefore suggest that LIF doesnot act through NO-mediated processes to induce changes inATP production. Our data suggest that the reduction in ATPand CrP content is related to downregulation of gene expres-sion and function of the ATP-synthase, and that these alter-ations are direct effects of LIF and not a consequence ofreduced contractile function.

The observed changes in CrP and ATP levels appear not tobe caused by alterations in CK enzymatic activity or CKisoform shift. CrP is the major high-energy phosphate re-serve compound in the heart and serves as a buffer forincreased demands of ATP [26]. Our results suggest that thereduced level of CrP is an indication of more CrP beingutilized to compensate for reducedATP production followingLIF treatment.

In the LIF treated myocytes we found a relatively largereduction in ATP-synthase enzymatic activity, but only amodest decrease in the a subunit mRNA. There are severalpossible explanations for the relatively large reduction inATP-synthase activity compared to the mRNA level. It ispossible that the protein levels of the a subunits, or the othersubunits in the ATP-synthase complex, were downregulatedmore than indicated by the mRNA measurements. Posttrans-lational modifications such as phosphorylation or nitrosyla-tion have to our knowledge not been described as a regulatorymechanism for the ATP-synthase. Another candidate forregulating the ATP-synthase activity is the inhibitor proteinIF1 [27]. This protein induces conformational changes uponbinding to the enzyme and thereby alters its activity.

There was no change in the NADH/NAD+ ratios in the LIFtreated cells. This suggests that there is not an accumulationof reducing equivalents, despite a reduced ATP-synthaseactivity following LIF treatment. Uncoupling of respiration,induced by uncoupling proteins, is able to maintainNADH/NAD+ ratios [28]. We suggest that such processesmay be induced in cardiomyocytes treated with LIF, butfurther experiments are necessary to investigate this in moredetail.

The reduction in ATP-synthase activity in LIF treated cellsis also relatively large compared to the reduction in ATP andCrP content. An increase in anaerobic energy metabolismfollowing LIF treatment contributes to ATP production andthus may partially compensate for the reduction in ATP-synthase activity. This notion is based on the significantincrease in glucose uptake and lactate content in LIF treatedcells, suggesting increased anaerobic metabolism. It has pre-viously been shown that anaerobic processes are able tomaintain the ATP content for 7 h in cardiomyocytes whenATP-synthase activity is inhibited [29]. In our study, it is thuslikely that anaerobic processes are able to partially compen-sate for the large reduction in ATP-synthase activity, but notcapable to maintain normal ATP levels following 48 h of LIFtreatment.

Although the ATP and CrP content do not directly corre-late with cardiac work, depletion of high-energy phosphates

together with reduced ATP-synthase activity in LIF treatedcells suggest a reduced ability to produce energy. A tightrelationship between the ability to produce energy and car-diac work has been described [30]. This notion is supportedby a study investigating ATP production and cardiac workwhen the ATP-synthase was inhibited by oligomycin [31].The authors found that oligomycin reduced contractile func-tion in an isolated rat heart model. However, the mechanismfor the adaptive reduction in contractile function resultingfrom reduced ability to produce high-energy phosphates ispoorly understood [30].

We also observed an increase in glucose uptake withoutany change in glucose oxidation in the LIF treated cells. TheLIF-mediated glucose uptake was not significantly differentfrom that mediated by insulin. These results are in agreementwith a recent report showing that LIF increased the glucoseuptake and the expression of the glucose transporterGLUT1 in isolated neonatal cardiomyocytes [32]. LIF treat-ment also increased the production of lactate following 48 hin culture. Since glucose oxidation did not increase, it islikely that glucose is shuttled to lactate production instead ofbeing oxidized. Increased lactate levels have been shown todecrease contractility in cardiomyocytes [33]. A possibleconsequence of elevated lactate and proton levels is redirec-tion of cellular ATP to maintain ion homeostasis at the ex-pense of contractile work [34]. Thus, increased lactate levelsmay partially be responsible for the reduced contractile func-tion of the LIF treated cardiomyocytes in our study.

LIF induced changes in lipid metabolism by increasingb-oxidation of oleic acid, but without increasing the com-plete oxidation to CO2. One possible explanation for thisfinding would be increased b-oxidation in the peroxisomes.However, we found the increase to be of mitochondrial ori-gin. It is then of interest why this does not cause an increasedformation of labeled CO2 from oleic acid and to understandthe fate of the incompletely oxidized fatty acids. One possi-bility is accumulation of acetyl-CoA, free acetate or citricacid cycle intermediates. However, since we measuredb-oxidation as ASM, we were not able to detect the level ofeach intermediate product separately. Another possibility isthat acetyl-CoA is shuttled to steroidogenesis or other path-ways, instead of being oxidized to CO2. Support for thisnotion is found in a recent report showing that LIF increasedsteroidogenesis in an adrenal carcinoma cell line [35]. Asimilar effect may therefore explain why the increase inb-oxidation does not cause more oleic acid to be oxidized toCO2 in cells treated with LIF. However, further studies arenecessary to determine the fate of the incompletely oxidizedoleic acid.

Incomplete oxidation of lipids and changes in lipid me-tabolism such as accumulation of triacylglycerol (TAG) oracyl-CoA may impair cell function [36]. However, our re-sults showed no changes in distribution of oleic acid to TAG.By measuring free oleic acid in the cells we also have anindirect measure of the acyl-CoA level, but we detected no

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differences following LIF treatment. Cell damage may alsobe induced by increased peroxisomal b-oxidation of lipidsdue to production of hydrogen peroxide and reactive oxygenspecies. However, our data showed that the increase inb-oxidation was of mitochondrial origin.

The mechanisms involved during transition to heart fail-ure are not known. Results of the present study suggest thatstimulation of the gp130/LIF receptor may contribute todevelopment of contractile dysfunction and changes in en-ergy metabolism. Myocardial failure, in the setting of hemo-dynamic overload, may be related to an inability of theenergy-producing system (i.e. mitochondria) to keep pacewith the needs of the contractile apparatus [10]. A shift insubstrate utilization from fatty acids to glucose and an in-crease in lactate levels have also been observed in failinghearts [11,34]. Taken together, the effects of LIF on themitochondrial ATP-synthase, glucose metabolism and lac-tate production observed in the present study suggest that LIFmay in part be responsible for the changes in energy metabo-lism in failing hearts.

We conclude that 48 h treatment of adult isolated cardi-omyocytes with LIF induces contractile dysfunction andaffects key enzymes and pathways for energy production.Some of these metabolic changes may be partially respon-sible for the contractile dysfunction in LIF treated cells.

Acknowledgements

This study was supported by The Norwegian Council forCardiovascular Diseases and Anders Jahre’s Fund for thePromotion of Science. We thank Morten Eriksen and LineSolberg for animal care. Jon Arne Birkeland, Unni L. Hen-riksen, Fredrik Swift and Tævje A. Strømme are acknowl-edged for technical assistance.

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