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European Journal of Heart Failure 2003 5(2):137-145; doi:10.1016/S1388-9842(02)00236-2
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© 2002 European Society of Cardiology

Leukemia inhibitory factor is augmented in the heart in experimental heart failure

Michihisa Jougasakia,b,*, Hanna Leskinenb, Amy M. Larsenb, Alessandro Cataliottib, Horng H. Chenb and John C. Burnett, Jr.b

a Institute for Clinical Research, National Hospital Kyushu Cardiovascular Center 8-1 Shiroyama-cho, Kagoshima 892-0853, Japan
b Cardiorenal Research Laboratory, Division of Cardiovascular Diseases, Mayo Clinic and Foundation Rochester, MN, USA

* Corresponding author. Tel.: +81-99-223-1151; fax: +81-99-226-9246 E-mail address: michi{at}qjun.hosp.go.jp


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Leukemia inhibitory factor (LIF) is a pleiotropic cytokine that induces cardiac myocyte hypertrophy through the signal transducing molecule, glycoprotein 130. To date, localization of LIF in the heart and regulation of cardiac LIF expression in congestive heart failure (CHF) remain undefined. The present study investigates the potential activation of LIF expression in the failing canine heart that was produced by progressive rapid ventricular pacing. Immunohistochemistry for LIF revealed that LIF immunoreactivity was present in the atrial and ventricular myocytes of the normal heart and was markedly increased in the failing heart as compared to the normal heart. Northern blot analysis demonstrated that cardiac LIF mRNA was increased in both atrium and ventricle in CHF as compared to the normal heart (P<0.01). Linear regression analysis revealed a positive correlation between atrial LIF mRNA and atrial pressure (r=0.87, P<0.001 in right atrium and r=0.86, P<0.001 in left atrium). Positive correlations between left ventricular LIF mRNA and left ventricular dimensions (r=0.91, P<0.0001 in end-systolic diameter; r=0.86, P<0.001 in end-diastolic diameter), and an inverse correlation between left ventricular LIF mRNA and left ventricular ejection fraction (EF) were observed (r=–0.93, P<0.0001). There was a positive correlation between left ventricular LIF mRNA and left ventricular mass index (LVMI) (r=0.85, P<0.001). The present study demonstrates that cardiac LIF immunoreactivity and its gene expression are increased in a canine model of experimental CHF and suggests a potential role for LIF in the pathophysiology of CHF.

Key Words: Cytokines • Gene expression • Heart failure • Congestive • Immunohistochemistry • Northern blot analysis

Received December 21, 2001; Revised June 18, 2002; Accepted October 18, 2002


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Leukemia inhibitory factor (LIF) is a pleiotropic cytokine, originally discovered on the basis of its ability to induce macrophage differentiation of the murine myeloid leukemia cell line M1 [1]. Subsequently, studies have elucidated that LIF has multiple biological actions on a variety of cell lines and tissues including haematopoietic cells, embryonal cells, hepatocytes, adipose tissues, bone and neurons [2,3]. LIF activates ras-dependent mitogen activated protein (MAP) kinase and Janus kinase-signal transducers and activators of the transcription (Jak-STAT) cascade pathway through the signal transducing molecule glycoprotein (gp) 130 [4]. Based on its structure and receptor signal-transducing molecule, LIF belongs to the interleukin-6-type cytokine family that includes interleukin-6, interleukin-11, oncostatin M, ciliary neurotrophic factor and cardiotrophin-1. Among these proinflammatory cytokines, LIF and cardiotrophin-1 are known to induce cardiac myocyte hypertrophy through gp130 in neonatal cardiac myocytes [5,6]. Recent studies using cultured neonatal rat cardiac myocytes have demonstrated that mechanical stretch augments gene expression of the interleukin-6-type cytokines such as LIF, cardiotrophin-1 and interleukin-6 [7].

Accumulating evidence has revealed that proinflammatory cytokines such as interleukin-6 are increased in patients with congestive heart failure (CHF) [8,9]. Soluble gp130, a circulating form of gp130 without signal transducing properties, is increased in human CHF [10]. Previous investigations have demonstrated that up-regulation of gp130 is caused by enhanced interleukin-6 stimulation [11]. Cardiotrophin-1 gene expression is significantly increased in the ventricular myocardium of 12-week-old spontaneously hypertensive-stroke prone rats at a stage of established hypertension [12]. Other investigators have also reported that ventricular cardiotrophin-1 and its receptor gp130 are up-regulated after myocardial infarction in rats [13]. We have recently demonstrated that cardiotrophin-1 is augmented in the heart in an experimental canine model of pacing-induced CHF [14]. Activation of several kinds of proinflammatory cytokines and associated receptors contributes to the progression of CHF once left ventricular dysfunction ensues [15]. Therefore, there are possibilities that other proinflammatory cytokines are augmented in CHF and play an important role in the pathophysiology of CHF. To date, the role of cardiac LIF during the progression of CHF remains unknown. We hypothesized that cardiac LIF expression is also enhanced in the failing heart.

We have established a canine model of experimental CHF produced by progressive rapid ventricular pacing for 38 days, which closely mimics human dilated cardiomyopathy with cardiac hypertrophy [16]. This model was characterized by decreases in left ventricular ejection fraction (EF), arterial blood pressure and cardiac output with increases in cardiac filling pressures, systemic vascular resistance and left ventricular mass index (LVMI). The present study was designed to investigate cardiac LIF immunoreactivity and its gene expression in this model of experimental CHF. In the present study, we compared LIF immunoreactivity and its gene expression in the failing heart to those in the normal heart by immunohistochemistry and Northern blot analysis, and demonstrated that cardiac LIF immunoreactivity and its gene expression are increased in a canine model of experimental CHF. We also showed not only positive correlations between atrial LIF gene expression and atrial pressure, but also relationships of ventricular LIF gene expression to cardiac dimension, function and hypertrophy.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Animal model of experimental CHF
Eleven male mongrel dogs weighing 19–24 kg were examined in the present study. Six dogs underwent implantation of a programmable cardiac pacemaker (Medtronic, Minneapolis, MN). Under pentobarbital sodium anesthesia (30 mg/kg, intravenous) and artificial ventilation (Harvard respirator, Harvard Apparatus, Millis, MA) with 5 l/min supplemental oxygen, left lateral thoracotomy and pericardiotomy were performed. The heart was exposed, and a screw-in epicardial pacemaker lead was implanted into the right ventricle. The pacemaker generator was implanted subcutaneously into the left chest wall and connected to the pacemaker lead. In addition, these dogs underwent implantation of a chronic femoral artery catheter (Model GPV Vascular-Access Port, Access Technologies, Skokie, IL) via the left femoral artery and subcutaneously connected to a port above the left upper hind limb. All dogs were allowed to recover for at least 10 days after surgery.

Experimental CHF was produced by progressive rapid ventricular pacing as reported previously [16,17]. After recovery from the surgery, the pacemaker was started with a stepwise increase of stimulation frequencies over 38 days. During the first 10 days, the dogs were paced at 180 beats per minute (bpm), and the pacing rate was increased weekly to 200, 210, 220 and 240 bpm to produce overt CHF with associated ventricular hypertrophy. All pacemakers were checked at the time of programming, then weekly and at the day of sacrifice for proper pacing. At baseline before pacing and after being paced at 240 bpm for 7 days, a 2D guided M-mode echocardiogram was obtained. Mean arterial blood pressure in the conscious state was measured via the femoral artery catheter in all dogs. Right atrial pressure, pulmonary artery pressure, pulmonary capillary wedge pressure and cardiac output were measured via a flow-directed balloon-tip catheter (American Edwards Laboratories, model 93131-7F, Anasco, PR) in the conscious state at baseline before pacing and at the end of the pacing at 240 bpm. Cardiac output was measured by thermodilution method using Cardiac Output Model 9520-A computer (Edwards Laboratories, Santa Ana, CA) in triplicate and averaged. Systemic vascular resistance and pulmonary vascular resistance were calculated according to the standard formula. At the end of the pacing at 240 bpm, the dogs were sacrificed and the cardiac tissues were rapidly harvested (Sleepaway euthanasia solution i.v., Fort Dodge Laboratories Inc., Fort Dodge, IA). Five age-matched normal dogs served as cardiac tissue donors for the normal control group. This study was approved by the Institutional Animal Care and Use Committee of the Mayo Clinic and conducted in accordance with the Animal Welfare Act.

2.2. Preparation for cardiac tissue analysis
Cardiac tissue analysis was performed on the atrial and ventricular myocardium. Atrial tissues were obtained from the appendages and free walls of both left and right atrium. Left ventricle was weighed for the calculation of LVMI (left ventricular weight (in grams) divided by body weight (in kilograms)). Full thickness sections of ventricular myocardium were obtained from the middle third of the free wall of both left and right ventricle. Each tissue was divided into two parts; one was frozen in liquid nitrogen and stored at –80 °C for Northern blot analysis, and the other was immediately fixed with 10% buffered formalin and embedded in paraffin. Paraffin sections were cut at a thickness of 5 µm and mounted on slide glasses treated with silica and used for immunohistochemistry.

2.3. Immunohistochemistry
Atrial and ventricular tissues were obtained from the normal dogs (n=5) and CHF dogs (n=6). Immunohistochemical study was performed using the indirect immunoperoxidase method as described previously [17,18]. In brief, the sections were incubated at 60 °C for 3 h and deparaffinized with xylene and graded concentrations of ethanol. To block the activity of endogenous peroxidase, the slides were incubated with 0.6% hydrogen peroxide in methanol for 20 min at room temperature. After washing, the sections were subsequently incubated with 5% horse serum (Vector Laboratories, Burlingame, CA) for 10 min at room temperature to reduce nonspecific background staining. The sections were then incubated with goat polyclonal anti-human LIF antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:400 in humidified chambers for 24 h at room temperature. All slides were incubated for 30 min with second antibody-horseradish peroxidase conjugate (Tago, Burlingame, CA) at a dilution of 1:100. The final reaction was achieved by incubating the sections with freshly prepared reagent containing 3-amino-9-ethylcarbazole (Sigma, St. Louis, MO) dissolved in dimethylformamide and sodium acetate. The sections were counterstained with hematoxylin, mounted, and reviewed with an Olympus microscope. Three trained independent observers reviewed these sections without any prior knowledge about the presence and absence of CHF. The staining intensity of LIF immunoreactivity was assessed by microscopic examination and semiquantitatively evaluated from 0 to 4 (0, absence of any staining of LIF; 1, minimal intensity; 2, mild intensity; 3, moderate intensity; and 4, maximal intensity).

To examine the immunohistochemical specificity of the reaction between antibody and tissue, absorption testing was performed. The anti-LIF antibody was preincubated with human recombinant LIF (Santa Cruz Biotechnology) overnight. After centrifugation at 4500 rpm for 10 min, the supernatant was used instead of primary antibody. The specificity was further confirmed by substitution of non-immune horse serum (NHS, Vector Laboratories) or phosphate buffered saline (PBS) for primary antibody.

2.4. Isolation of mRNA
Messenger RNA was isolated from the canine cardiac tissues using the FastTrackTM 2.0 kit (Invitrogen, Carlsbad, CA) as previously described [14]. In brief, canine cardiac tissue was initially lysed in detergent-based buffer containing RNAse/Protein Degrader, incubated at 45 °C and applied directly to Oligo (dT) cellulose for adsorption. DNA, degraded proteins and cellular debris were washed with a high salt buffer (Binding buffer). Non-polyadenylated RNA was washed with a low salt buffer (Low salt buffer), and the polyadenylated RNA was then eluted in the absence of salt (Elution buffer). Purity and quality of the isolated mRNA was assessed by reading optical densities at 260 and 280 nm and by electrophoresis in 1.2% denaturing agarose gel.

2.5. Probe for canine LIF
The cDNA sequence for canine LIF has not been reported. To obtain a specific probe for canine LIF, reverse transcription-polymerase chain reaction (RT-PCR) was performed using the degenerated primers. First-strand cDNA was synthesized from 1 µg of canine spleen mRNA using oligo (dT) primer and Moloney murine leukemia virus reverse transcriptase (Perkin–Elmer, Norwalk, CT). The oligonucleotide primers were synthesized using an Applied Biosystems 394 DNA/RNA synthesizer (Foster, CA). For amplification of canine LIF cDNA fragment, the following degenerate primers were selected based on the mammalian LIF cDNA sequence [19–22]: sense (human LIF: 106–126), 5'-TCT(C/G)CACTGGAAACA(C/T)GGGGC-3'; antisense (human LIF: 428–448), 5'-GGGGTT(C/G)AGGA(C/T)CTTCTGGTC-3', yielding a predicted product of 343 bp. A Perkin–Elmer 2400 Thermocycler was used to amplify the samples. PCR cycling parameters were as follows: denaturation at 94 °C for 3 min followed by denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min and extension at 72 °C for 1 min. Thirty cycles were performed with a final extension time for 7 min. The PCR products were cloned into pCRTM TA cloning vector (Invitrogen, San Diego, CA) and sequenced by ABI Prism dideoxy chain termination method using Applied Biosystems 377 Automated DNA Sequencer. The clone contained a 343-bp fragment, which encoded the deduced structure of canine LIF (GenBank Accession Number: AF512028 [GenBank] ).

2.6. Northern blot analysis
Atrial and ventricular tissues were obtained from the normal dogs (n=5) and the CHF dogs (n=6). For Northern blot analysis, 4 µg of mRNA isolated from the canine tissues were loaded on a 1.2% agarose formaldehyde gel and electrophoresed for 3 h at 70 V. The gel was transferred downward onto a nylon membrane (Maximum Strength Nytran Membrane, Schleicher & Schuell, Keene, NH) overnight using TurboblotterTM (Schleicher & Schuell) and cross-linked with using UV StratalinkerTM (Stratagene, La Jolla, CA). Canine LIF cDNA probe was labeled with 32P-dATP by a random-priming labeling kit (Strip-EZTM DNA, Ambion Inc., Austin, TX) and purified using G-50 Quick Spin Columns (Roche Diagnostics Co., Indianapolis, IN). Labeled canine LIF cDNA probe was hybridized to the membranes at 68 °C overnight in Quick-hyb hybridization solution (Stratagene). The membranes were washed in 2x SSC, 0.1% SDS at 22 °C for 15 min, then in 0.2x SSC, 0.1% SDS at 22 °C for 15 min, and in 0.2x SSC, 0.1% SDS at 55 °C for 20 min. Autoradiography was performed with Kodak X-ray film at –80 °C overnight. To standardize loading conditions and mRNA transfer onto the membranes, blots were stripped and re-hybridized with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe.

2.7. Statistical analysis
Results of the values are expressed as mean±S.E.M. Statistical comparisons between the normal dogs and the CHF dogs were performed by Student's unpaired t-test. The correlation analysis was performed using linear regression analysis. Statistical significance was accepted for P value less than 0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Cardiovascular haemodynamic and echocardiographic data
Table 1 summarizes the cardiovascular haemodynamic and echocardiographic data in the normal and CHF dogs. Experimental CHF was characterized by decreases in mean arterial blood pressure (–12%), cardiac output (–53%) and left ventricular EF (–63%) with increases in cardiac filling pressures (right atrial pressure, +180%; pulmonary capillary wedge pressure, +130%) and systemic vascular resistance (+65%). Pulmonary vascular resistance tended to increase (+67%), although not significantly. Left ventricular dimensions significantly increased (+23% in left ventricular end-diastolic diameter and +68% in left ventricular end-systolic diameter). All six CHF dogs had pulmonary edema, pleural effusion and ascites. These results indicate that the CHF dogs had cardiac dilatation and dysfunction with signs of pulmonary and systemic congestion.


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  		Table 1 Cardiovascular haemodynamic and echocardiographic data in the normal and CHF dogs

 
3.2. Immunohistochemical staining for LIF
Fig. 1A illustrates representative immunohistochemical staining for LIF in the atrium and ventricle of the normal and failing canine heart. Immunohistochemical staining revealed faint positive LIF immunoreactivity in the atrial and ventricular myocytes in the normal heart. LIF immunoreactivity was increased in the atrial and ventricular myocytes of the failing heart as compared to the normal heart. The degrees of LIF immunoreactivity in two groups are shown in Fig. 1B. The grade of immunoreactivity was low in the normal heart (atrium, 0.5±0.3; ventricle, 0.3±0.2) and higher in the failing heart (atrium, 1.5±0.2, P<0.05 vs. normal group; ventricle, 1.2±0.1, P<0.05 vs. normal group). LIF immunoreactivity was observed in the cytoplasm of cardiac myocyte and was distributed widely in the peripheral cytoplasm. There was no evidence of immunoreactivity in the connective tissues. The sections treated with pre-absorbed antibody, NHS or PBS instead of primary antibody failed to show immunoreactivity in the cardiac myocytes.


Figure 1
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  		Fig. 1 (A) Immunohistochemical staining for LIF in the canine heart of the normal dogs and the CHF dogs. The atrial and ventricular sections were incubated with an anti-LIF antibody and then with horseradish-conjugated Fab fragment of anti-goat IgG. Control for immunohistochemistry is NHS. Original magnification 400x. (B) Bar graph of the degree of LIF immunoreactivity in the atrium and ventricle of the normal dogs and the CHF dogs. Both atrial and ventricular LIF immunoreactivities were greater in the CHF dogs than in the normal dogs. *P<0.05 vs. normal.

 
3.3. Northern blot analysis for LIF mRNA
Fig. 2A illustrates representative Northern blot analysis for LIF mRNA in the atrium and ventricle of the normal and failing canine heart. LIF gene expression was detected in both atrium and ventricle at the size of 4.2 kb. Both atrial and ventricular LIF mRNA were increased in the failing heart. The ratio of LIF mRNA to GAPDH mRNA increased 3.4 times in the failing atrium and 2.9 times in the failing ventricle as compared to the normal heart (Fig. 2B). Linear regression analysis revealed positive correlations between right atrial LIF mRNA and right atrial pressure (r=0.87, P<0.001, Fig. 3A) as well as between left atrial LIF mRNA and pulmonary capillary wedge pressure that represents left atrial pressure (r=0.86, P<0.001, Fig. 3B). Positive correlations between left ventricular LIF mRNA and left ventricular dimensions (r=0.91, P<0.0001 in end-systolic diameter; r=0.86, P<0.001 in end-diastolic diameter, Fig. 4A), and an inverse correlation between left ventricular LIF mRNA and left ventricular EF (r=–0.93, P<0.0001, Fig. 4B) were observed.


Figure 2
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  		Fig. 2 (A) Northern blot analysis for LIF in the atrium and ventricle of the normal dogs (N) and the CHF dogs. Four microgram of mRNA were isolated from the atrium and ventricle, transferred to a nylon membrane and hybridized with a canine LIF cDNA probe. Blots were stripped and re-hybridized with GAPDH cDNA probe. The expression of GAPDH mRNA was used as an internal control. Both atrial and ventricular LIF mRNA levels in the CHF dogs were increased as compared to those in the normal dogs. (B) Bar graph showing the results of densitometric analysis of Northern blotting in the normal dogs and the CHF dogs. Density of LIF mRNA blots was counted on a BioRad Fluor-S MultiImager and normalized to GAPDH mRNA level. GAPDH mRNA level was similar in almost all atrial and ventricular samples from dogs tested. Data are mean±S.E.M. for the LIF mRNA levels corrected by GAPDH mRNA levels and expressed as arbitrary units. The ratio of LIF mRNA to GAPDH mRNA in both atrium and ventricle of the CHF dogs was increased as compared to that of the normal dogs. *P<0.05 vs. Normal dogs.

 


Figure 3
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  		Fig. 3 (A) Graph showing correlation between right atrial pressure (RAP) and right atrial LIF gene expression. Right atrial LIF mRNA positively correlated with RAP (y=0.043X–0.015, r=0.87, P<0.001). (B) Graph showing correlation between pulmonary capillary wedge pressure (PCWP) and left atrial LIF gene expression. Left atrial LIF mRNA positively correlated with PCWP, which represents left atrial pressure (y=0.029X–0.11, r=0.86, P<0.001).

 


Figure 4
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  		Fig. 4 (A) Graph showing correlation between left ventricular dimension and left ventricular LIF gene expression. Left ventricular LIF mRNA positively correlated with left ventricular end-diastolic diameter (LVDd) (y=0.012X–0.389, r=0.86, P<0.001). (B) Graph showing correlation between left ventricular EF and left ventricular LIF gene expression. Left ventricular LIF mRNA inversely correlated with left ventricular EF (y=–0.003X+0.275, r=–0.93, P<0.0001). (C) Graph showing correlation between left ventricular LIF gene expression and LVMI. Left ventricular LIF mRNA positively correlated with LVMI (y=0.126X–0.443, r=0.85, P<0.001).

 
3.4. Left ventricular mass index
LVMI was 4.2±0.1 g/kg in the normal dogs and increased to 5.0±0.1 g/kg in the CHF dogs (P<0.05). There was a positive correlation between left ventricular LIF mRNA and LVMI (r=0.85, P<0.001) (Fig. 4C).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
In the present study, we report the localization of LIF immunoreactivity in the atrial and ventricular myocytes of the canine heart and its increased immunoreactivity in the failing heart. This study also demonstrates that gene expression of LIF is present in both atrial and ventricles of the canine heart, and that cardiac gene expression of LIF is enhanced in CHF. The alteration in immunoreactivity supports augmented production of LIF in CHF. Furthermore, linear regression analysis revealed that atrial LIF gene expression increases in accordance with atrial pressure, and that augmentation of ventricular LIF gene expression is associated with increases in ventricular dimension and hypertrophy and a decrease in cardiac function.

To date, the role of LIF in the heart has not been fully clarified. The present study demonstrates that LIF is increased in the failing heart and raises the possibility that LIF could contribute to the progression and pathophysiology of CHF. LIF elicits its biological actions through signal transducing molecule, gp130. Heterodimerization of gp130 and LIF receptor activates distinct signaling pathways, such as MAP kinase system and Jak-STAT cascade pathway [4]. Over-expression of the gp130 resulted in cardiac hypertrophy [23], and targeted disruption of gp130 led to a hypoplastic ventricular myocardium [24]. Ventricular gp130 knock-out mice have normal cardiac structure, but develop rapidly progressive dilated cardiomyopathy when subjected to pressure overload [25]. These studies suggest that cardiac gp130 plays an important role in cardiac hypertrophy in CHF. While the significance of increased endogenous LIF gene expression in the canine failing heart remains unknown, the strong relationship between left ventricular LIF gene expression and LVMI supports a possible role of ventricular LIF in the hypertrophic process in the failing heart in CHF. Further studies will be needed to determine if increased ventricular LIF production is the trigger for cardiac hypertrophy during the progression of CHF.

The mechanism of enhanced cardiac LIF immunoreactivity and its gene expression in CHF is not known. Circulating levels of cytokines such as interleukin-6 are increased in human CHF [8], and cardiac interleukin-6 and interleukin-6 receptor are up-regulated in advanced human CHF [9]. Recently, we have reported that cardiotrophin-1 which also belongs to the interleukin-6 cytokine family is augmented in the heart in experimental CHF in dogs [14]. A similar finding of up-regulation of cardiotrophin-1 was observed in the myocardial infarction model in rats [13]. These findings raise the possibility that the interleukin-6-type cytokines that produce cardiac hypertrophy are activated in CHF and play an important role in the pathophysiology of CHF. The present study provides direct evidence of enhanced cardiac production of LIF in an experimental model of CHF. Although synthesis and metabolism of LIF in the heart are not yet fully understood, like other neurohumoral factors activated in the failing heart [26], cardiac LIF expression may be augmented in association with haemodynamic changes and increased chamber wall stress. Recently, Pan et al. have reported that mechanical stretch augments gene expression of LIF in the neonatal rat cardiac myocytes in vitro [7]. Therefore, it is tempting to speculate that mechanical stretch also stimulates LIF production in the failing heart in vivo.

In the present study, LIF was more expressed in the atrium as compared to the ventricle in the failing heart. Although the reason for the different expression of LIF between atrium and ventricle is unknown, LIF might have a different role in the atrium as compared to the ventricle. Recent investigators have demonstrated higher LIF gene expression in the failing ventricle than in the non-failing ventricle in human [27]. However, they failed to show increased LIF gene expression in the failing human atrium. In the present study, we have demonstrated that both atrial and ventricular LIF gene expressions are augmented in the failing heart as compared to the normal heart. Although the discrepancy between the previous human study [27] and our present experimental study remains unknown, there are possibilities that cytokine levels in the human heart are modulated by various mechanical stress, neurohumoral factors and effective drug treatment.

The major objective of the present study was to quantitate LIF mRNA in the atrial and ventricular myocardium and to localize LIF immunoreactivity in the normal heart and the failing heart. To date, the cDNA encoding canine LIF has not been cloned. Therefore, we developed 343-bp canine LIF cDNA probe for Northern blot analysis using RT-PCR with degenerate primers for LIF (GenBank Accession Number: AF512028). This putative partial canine LIF probe had 92% homology to human LIF cDNA [19], 87% homology to bovine LIF cDNA [20], 85% homology to mouse LIF cDNA [21], and 83% homology to rat LIF cDNA [22]. Further cloning studies will be needed for complete sequence analysis of canine LIF cDNA and genomic DNA. In addition, we used anti-human LIF antibody to localize canine LIF immunoreactivity in the heart for the present immunohistochemical examination. As the LIF immunoreactivity in the heart was diminished by pre-absorbed antibody in the absorption test, immunoreactive substance in the canine heart is considered to be ‘canine LIF’.

In summary, the present study demonstrates that LIF immunoreactivity and its gene expression are augmented in both atrium and ventricle in a canine model of experimental CHF. We also report important correlations not only between atrial LIF mRNA and atrial pressure, but also between ventricular LIF mRNA and ventricular parameters of dimension, function and hypertrophy. This study strongly supports a potential role for LIF in the pathophysiology of CHF, and provides important new insights into another local myocardial cytokine activation in CHF.


   Acknowledgements
 
We acknowledge the technical assistance of Denise M. Heublein, Sharon M. Sandberg and Gail J. Harty. This work was supported by grants from American Heart Association of the Minnesota Affiliate (MN-97-GB-06), the National Institute of Health (HL 36634), National Kidney Foundation of the Upper Midwest, the Miami Heart Research Institute, the Bruce and Ruth Rappaport Program in Vascular Biology, and Mayo Foundation.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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