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Cardiovascular Research 2006 69(2):545-555; doi:10.1016/j.cardiores.2005.11.014
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Copyright © 2005, European Society of Cardiology

Changes in cardiac lipid metabolism during sepsis: The essential role of very low-density lipoprotein receptors

Lijing Jiaa, Masafumi Takahashia,*, Hajime Morimotoa, Sadao Takahashib, Atsushi Izawaa, Hirohiko Isea, Tadao Iwasakic, Hiroaki Hattoric, Kou-Juey Wud and Uichi Ikedaa

aDepartment of Organ Regeneration, Shinshu University Graduate School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan
bThird Department of Internal Medicine, Faculty of Medical Science, University of Fukui, Fukui, Japan
cDepartment of Advanced Medical Technology and Development, BML Inc., Saitama, Japan
dInstitute of Biochemistry, National Yang-Ming University, Taipei, Taiwan

* Corresponding author. Tel./fax: +81 263 37 3193/+81 63 37 2573. Email address: masafumi{at}sch.md.shinshu-u.ac.jp

Received 8 August 2005; revised 7 November 2005; accepted 9 November 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective: Sepsis accompanies myocardial dysfunction and dynamic alterations of cardiac metabolism. We have recently demonstrated that the very low-density lipoprotein receptor (VLDL-R), which is abundantly expressed in the heart, plays a key role in energy metabolism of the fasting heart. However, little is known about the function and regulation of the VLDL-R during sepsis. In the present study, we explored lipid accumulation and VLDL-R expression in the lipopolysaccharide (LPS)-stimulated heart in vivo and regulation of VLDL-R expression in vitro.

Methods and results: Electron microscopy and immunohistochemistry demonstrated that LPS significantly decreased both lipid accumulation and VLDL-R expression in the hearts of fasting mice. Treatment with LPS also downregulated VLDL-R in rat neonatal cardiac myocytes, and this downregulation was completely reversed by interleukin (IL)-1β receptor antagonist. IL-1β downregulated the expression of VLDL-R in a time- and dose-dependent manner and markedly reduced the uptake of DiI-labeled β-VLDL but not DiI-labeled low-density lipoprotein (LDL). Use of specific pharmacologic inhibitors and short interference RNA revealed that Hsp90 was required for IL-1β to downregulate VLDL-R expression.

Conclusions: These findings suggest that IL-1β is a principle mediator of changes in cardiac lipid and energy metabolism during sepsis through the downregulation of myocardial VLDL-R expression.

KEYWORDS Cytokines; Inflammation; Lipoproteins; Myocytes; Receptors


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Sepsis has been defined as the systemic inflammatory response to infection. It is now accepted that human septic shock is accompanied by myocardial dysfunction and dynamic alterations of cardiac metabolism [1–5]. A number of myocardial depressant factors in human septic patients have been proposed; however, the identification of the responsible molecules remains unclear. Kumar et al. [6] were the first to demonstrate that the negative effects of sepsis on myocardial function were mediated, at least in part, through inflammatory cytokines such as tumor necrosis factor{alpha} (TNF{alpha}) and interleukin-1β (IL-1β). Increased serum TNF{alpha} levels in patients with septic shock have been demonstrated and the cardiovascular effects of TNF{alpha} and clinical septic shock are similar. IL-1β induces changes in cardiovascular function similar to TNF{alpha} [7] and is increased in both human and experimental models of sepsis and septic shock [8]. When cardiac myocytes are exposed to IL-1β, contractility is depressed [9] and immunoabsorption of IL-1β partially neutralizes the depressant activity of human septic serum [6]. Administration of an IL-1β antagonist attenuates the hemodynamic and metabolic manifestations of septic shock [10]. Although these studies have provided a crucial role for inflammatory cytokines in sepsis-induced myocardial dysfunction, the cellular and molecular mechanisms have not been fully elucidated.

The receptor for very low-density lipoprotein (VLDL) was cloned and characterized in 1992 [11] and has been shown to be distinctly different from the low-density lipoprotein (LDL) receptor (LDL-R) with respect to its ligand-binding properties and tissue distribution. The VLDL receptor (VLDL-R) binds specifically to apolipoprotein E-containing particles such as VLDL, intermediate density lipoprotein (IDL), and β-migrating VLDL (β-VLDL), but does not bind LDL. We previously demonstrated that the VLDL-R is expressed in human macrophages and plays a crucial role in macrophage foam cell formation in the development of atherosclerosis [12]. Although the VLDL-R is highly expressed in the heart, little is known of its influence on cardiac function or metabolism. We recently found that the expression of VLDL-R in the heart is increased with growth and fasting, and associated with a switch in energy substrate (from glucose to fatty acids) [13]. Furthermore, recent evidence suggests that VLDL might play a regulatory role in heart lipid metabolism through its receptor-mediated mechanism [14]. We therefore hypothesized that the VLDL-R plays a role in myocardial dysfunction during septic shock through changes in cardiac lipid metabolism. In the present study, we explored lipid accumulation and VLDL-R expression in the heart during septic shock induced by lipopolysaccharide (LPS) in vivo and the regulation of VLDL-R expression in vitro. The findings obtained from this study provide new insight into the pathogenesis of sepsis-induced myocardial dysfunction and lipid metabolism.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1 Experimental animals and protocols
Balb/C mice (male, 12-week-old) and pregnant Sprague–Dawley rats were purchased from Japan SLC Inc. (Tokyo, Japan). They had free access to standard chow and water, and were maintained on a 12-h light and dark cycle. All experiments in this study were performed in accordance with the Shinshu University Guide for Laboratory Animals, which conforms to NIH Guidelines. For fasting experiments, mice had food, but not water, withdrawn at 20:00, and received intraperitoneal injection of LPS (10 mg/kg) (fasting and control mice, n=3–6). The body weights were markedly decreased (approximately 20% decrease) during 24 h of fasting. The mice were sacrificed after 36 h of fasting, and heart samples were prepared for electron microscopy, immunostaining, and immunoblotting.

2.2 Cell cultures and reagents
Neonatal cardiac myocytes were prepared from ventricles of 1-day-old Sprague–Dawley rats as described previously [15]. Cell suspensions were washed with Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and centrifuged at 500 x g for 5 min. The centrifuged cells were then resuspended in 10% FBS-containing DMEM. For selective enrichment of cardiac myocytes, the dissociated cells were preplated for 60 min, during which time non-cardiac myocytes readily attached to the bottom of the culture dish. Using this method, we routinely obtained enriched cultures containing >90% myocytes, as assayed by immunofluorescence staining with an anti-myosin heavy chain antibody.

The expression plasmid for short interference RNA (siRNA) of heat shock protein 90 (Hsp90: Hsp86{alpha} for mouse/rat homolog: pSUPERHspiR) was generated as described previously [16]. The green fluorescent protein (GFP) expressing plasmid pEGFP-C1 was obtained from BD Bioscience Clontech (Mountain View, CA). Polyclonal antibodies against the rat VLDL receptor (VR2) were generated as described previously [12]. Anti-cardiac troponin I (cTnI) monoclonal antibody was obtained from Biogenesis (England, UK). Anti-Hsp90 monoclonal antibody was purchased from Stressgen (Victoria, Canada) and BD Transduction Laboratories (San Jose, CA). IL-1β, TNF{alpha}, and interferon-{gamma} (IFN-{gamma}) were obtained from Genzyme/Techne (Minneapolis, MN). Human endothelin-1 (ET-1) was purchased from Peptide Institute Inc. (Tokyo, Japan). The IL-1 receptor antagonist (IL-1RA) was purchased from Pepro Tech Inc. (Rocky Hill, NJ). Herbimycin A (HA), geldanamycin (GA), genistein, radicicol, sodium nitroprusside (SNP), and NG-monomethyl-L-arginine (L-NMMA) were purchased from Calbiochem (San Diego, CA). The remaining reagents including LPS (0111:B4) and angiotensin II (AII) were obtained from Sigma unless otherwise specified.

2.3 Transmission electron microscopy
The left ventricles of mice were cut into 1-mm thick and fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) at 4 °C for 24 h; tissues were washed with ice-cold phosphate buffer (0.1 M, pH 7.4). For transmission electron microscopy, tissues were post-fixed in 1% osmium tetroxide at 4 °C for 1 h, and then dehydrated in a graded series of ethanol, substituted with propylene oxide, and embedded in Epon resin (Epok 812, Oken, Tokyo, Japan). Ultrathin sections were stained with uranyl acetate and lead citrate and observed with a conventional transmission electron microscope (JEM-1200EX, JOEL, Tokyo, Japan) at an accelerating voltage of 80-kV. Photographs were taken at x 3000.

2.4 Transfection
Gene transfection of cardiac myocytes with pSUPER–HspiR and pEGFP-C1 was performed using a Rat Cardiomyocyte-Neonatal Nucleofector Kit (Amaxa Biosystems, Cologne, Germany), according to the manufacturer's instruction. Briefly, freshly isolated cardiac myocytes (1 x 106 cells) were suspended in the specified electroporation buffer, and 3.6 µg of pSUPERHspiR and 0.4 µg of pEGFP-C1 were added subsequently. After the mixed solution was added to the special Nucleofector device (Amaxa Biosystems) for gene transfection, the transfected cells were cultured on the cover slips in eight culture plates (1 x 105 cells/well, Lab-Tek chamber slide, Nunc, IN).

2.5 Immunostaining
For immunostaining for VLDL-R in cardiac myocytes, the cells were cultured on the cover slips in eight culture plates (Lab-Tek chamber slide) for 4 days, and treated with or without IL-1β for 48 h. The cells were washed with phsoshate-buffered saline (PBS), fixed with 4% paraformaldehyde in PBS for 10 min, and treated with 3% hydrogen peroxide for 10 min to block endogenous peroxidase activity. Blocking was with 5% bovine serum albumin (BSA) for 15 min, incubated with anti-VLDL-R antibody (VR2: 1/600) for 1 h, followed by incubation with peroxidase-conjugated anti-rabbit IgG (Zymed Laboratories Inc., San Francisco, CA) for 45 min. The immunoreactive cells were detected with 3,3'-diaminobenzidine tetrahydrochloride (DAB Substrate Kit: Vector Laboratories Inc., Burlingame, CA).

For immunofluorescence double staining, the cells that were transfected with the corresponding plasmids were cultured on the cover slips in eight culture plates (Lab-Tek chamber slide) for 4 days, and treated with or without IL-1β for 48 h. The cells were fixed and blocked as described above, and incubated with primary antibodies against Hsp90 (2.5 µg/mL), VLDL-R (VR2: 1/600), and GFP (polyclonal: 1/500, monoclonal: 2 µg/mL) for 1 h. The secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) were used as follows: goat anti-mouse IgG conjugated with Cy3 for Hsp90, goat anti-mouse IgG conjugated with FITC for GFP (monoclonal), goat anti-rabbit IgG conjugated with Cy3 for VLDL-R, and goat anti-rabbit IgG conjugated with FITC for GFP (polyclonal). The cells were incubated with DAPI (Wako Pure Chemical Ltd., Osaka, Japan) for nucleic acid staining. After washing, the cells were mounted and observed using confocal laser scanning microscopy (Leica TCS-SP2 AOBS spectral, Heidelberg, Germany). No signals were detected when normal goat serum or irrelevant IgG was used instead of the primary antibody as a negative control.

2.6 Lipoprotein preparation and immunofluorescence detection
β-VLDL and LDL were prepared and labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI) (Molecular Probes, Inc., Eugene, OR) as described previously [17]. To evaluate the uptake of DiI-lipoproteins, cells were treated with or without 10 ng/mL IL-1β or 1000 U/mL IFN-{gamma} for 24 h, washed twice with PBS, and then incubated with 5% lipoprotein-depleted serum (LPDS)/DMEM containing 10 µg/mL DiI-labeled β-VLDL (DiI-β-VLDL) or 10 µg/mL DiI-labeled LDL (DiI-LDL) for 3 h at 37 °C. The cells were washed with 2% BSA/PBS and fixed with 4% formalin in 0.3 M phosphate buffer (pH 7.4). The uptake of DiI-lipoproteins was detected by fluorescence microscopy (BX-51, Olympus, Tokyo, Japan).

2.7 Immunoblotting
Immunoblotting was performed as described previously [18]. Briefly, cells were washed with ice-cold PBS and then lysed in RIPA buffer (25 mM Tris–HCl, pH 7.5, 2.5 mM EDTA, 10 mM sodium pyrophosphate, 50 mM NaF, 137 mM NaCl, 100 µM Na3VO4, 1% Triton X-100, 10% glycerol, 1% deoxycholic acid, 0.1% SDS, and fresh 0.1 mM phenylmethyl sulfonyl fluoride (PMSF) and 10 µg/mL of leupeptin). The cell lysates were prepared by scraping, sonication, and centrifugation. Cellular protein concentrations were determined by the DC protein assay (Bio-Rad). Cell lysates were subjected to SDS-PAGE under reducing conditions, and the protein bands then transferred to a nitrocellulose membrane. The membrane was blocked for 2 h at room temperature with 5% skim milk, and then incubated for 1 h at room temperature with the primary antibodies, followed by incubation for 1 h with the secondary antibody, conjugated horseradish peroxidase. Immunoreactive bands were visualized by an enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech UK Ltd., Buckinghamshire, UK), with the level of protein expression being quantified using NIH image 1.63 software.

2.8 Immunoprecipitation
Immunoprecipitation were performed as described previously [17]. Briefly, the cells were washed with ice-cold PBS and lysed in gentle lysis buffer (25 mM Tris–HCl, pH 7.5, 2.5 mM EDTA, 10 mM sodium pyrophosphate, 50 mM NaF, 137 mM NaCl, 100 µM Na3VO4, 1% Triton X-100, 10% glycerol, and fresh 0.1 mM PMSF and 10 µg/mL of leupeptin). Immunoprecipitation was carried out in the presence of the primary antibody for 2 h at 4 °C, followed by overnight incubation with a protein A-Sepharose slurry. Following washing, the immunoprecipitates were boiled in sample buffer, separated by SDS-PAGE, transferred, and immunoblotted.

2.9 Realtime RT-PCR analysis
Total cellular RNA was prepared from cardiac myocytes by the guanidine isothiocyanate–cesium chloride (GITC–CsCl) method using RNAeasy (Invitrogen) as described previously [15]. Realtime RT-PCR analysis was performed to detect the mRNA expression of inducible nitric oxide synthase (iNOS) by using the ABI Prism 7000 system (PE Applied Biosystems, Inc.); the primers and PCR conditions for VLDL-R and iNOS have been described previously [17,19]. The expression levels of each target gene were normalized by subtracting the corresponding β-actin threshold cycle (CT) values by using the {Delta}{Delta} CT comparative method.

2.10 Statistical analysis
Data are expressed as the mean ± SD. For comparisons between multiple groups, we determined the significance of the difference between group means by one-way analysis of variance (ANOVA) or an unpaired t test. All analysis was performed using StatView software (Abacus Concepts, Inc., Berkeley, CA). Differences with p values<0.05 were considered to be statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1 LPS decreases lipid accumulation and VLDL-R expression in the fasting heart
Since we previously found that VLDL-R expression was abundantly expressed in the myocardium and increased in the fasting heart [13], we first examined the effect of LPS administration on lipid accumulation and VLDL-R expression in the fasting mouse heart. Electron microscopy revealed that lipid accumulation was increased in the fasting heart compared to the normal or LPS-treated heart, and this fasting-induced lipid accumulation was decreased by the administration of LPS (Fig. 1A). Immunohistochemical and Western blot analyses demonstrated that a significant increase of VLDL-R expression was observed in the fasting heart and this expression was significantly attenuated by the administration of LPS (Fig. 1A, C, and D). Although treatment with LPS alone showed no changes in lipid accumulation, VLDL-R expression was decreased by this treatment. To identify the cell types that expressed VLDL-R, double immunohistological staining using antibodies against VLDL-R and the cardiac specific marker cTnI was performed. Co-expression of VLDL-R and cTnI in the fasting heart was clearly observed (Fig. 1B), indicating that cardiac myocytes express VLDL-R.


Figure 1
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  		Fig. 1 LPS decreases lipid accumulation and VLDL-R expression in the fasting heart. Experimental protocol for fasting and LPS administration is described in the "Materials and methods" section. (A) Representative electron microscopic views and immunostaining of the VLDL-R in left ventricular tissue from a control mouse (Cont, n=6), a mouse treated with LPS (LPS, n=3), fasting mouse (Fasting, n=6), and a fasting mouse treated with LPS (Fasting+LPS, n=6). (B) Representative immunostaining of cTnI and VLDL-R in the fasting mouse heart, and merged images are shown. Bar shows 100 µm. (C) Cell lysates were prepared from the left ventricular tissue and analyzed by immunoblotting with antibodies against the VLDL-R or β-actin. (D) The bar graph shows the relative expression levels of the VLDL-R quantified by NIH image 1.63 software. Results are expressed as means ± SD (n=3–6). *p<0.05 and **p<0.01.

 
3.2 IL-1β is a key mediator for LPS-downregulated VLDL-R expression in cardiac myocytes
To investigate the mechanisms of LPS-attenuated lipid accumulation and VLDL-R expression in the myocardium, we used rat neonatal cultured cardiac myocytes in vitro. As expected, treatment with LPS downregulated VLDL-R expression in cardiac myocytes in a dose- and time-dependent manner (Fig. 2A and B). There were no significant changes in protein levels of β-actin that served as an internal control for protein loading.


Figure 2
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  		Fig. 2 IL-1β is a principle mediator for LPS-downregulated VLDL-R expression in cardiac myocytes. (A) Cardiac myocytes were treated with 100 ng/mL LPS for the indicated periods. (B) Cells were treated for 48 h with LPS at the concentrations indicated. Results are expressed as the mean ± SD (n=3). *p<0.05, **p<0.01, and ***p<0.001. (C) Cells were treated with either 10 ng/mL IL-1β, 10 ng/mL TNF{alpha}, 1000 U/mL IFN-{gamma}, 100 nM AII, or 100 nM ET-1 for 48 h. (D) Cells were pretreated for 1 h with IL-1RA at the concentrations indicated, and then were treated with 100 ng/mL LPS for 48 h. (E) Cells were pretreated for 1 h with 100 µM SNP or 1 mM L-NMMA (L-NM), and then were treated with 10 ng/mL IL-1β for 48 h. Cell lysates were then prepared and analyzed by immunoblotting with antibodies against the VLDL-R or β-actin. The results are representative of three independent experiments.

 
Administration of LPS induces production of proinflammatory cytokines such as IL-1β, TNF{alpha}, and IFN-{gamma} in the myocardium [5]. To investigate the key factor for LPS-induced VLDL-R downregulation, cells were treated with recombinant IL-1β, TNF{alpha}, IFN-{gamma}, AII, and ET-1, and then analyzed for VLDL-R expression. Treatment with IL-1β, but not TNF{alpha}, IFN-{gamma}, AII, and ET-1, completely downregulated VLDL-R expression (Fig. 2C). Furthermore, inhibition of the IL-1-signaling pathway by IL-1RA prevented the downregulation of VLDL-R expression in response to LPS, and this inhibitory effect of IL-1RA was dose dependent (0.1–1 µg/mL) (Fig. 2D).

We previously reported that treatment with IL-1β markedly stimulated nitric oxide (NO) production in cardiac myocytes [20]. In addition, since NO is one of the important factors for LPS-induced myocardial dysfunction [5], we tested the effect of the NO donor, SNP, and the NO inhibitor, L-NMMA, on VLDL-R expression. Pretreatments with these agents had no effect on VLDL-R expression in unstimulated and IL-1β-stimulated cardiac myocytes (Fig. 2E). These results indicate that IL-1β is a key mediator of the effect of LPS on VLDL-R expression in the myocardium.

3.3 IL-1β downregulates VLDL-R expression in cardiac myocytes
We confirmed the inhibitory effect of IL-1β on VLDL-R expression in cardiac myocytes by immunocytochemical analysis (Fig. 3A) and further examined the time- and dose-dependent effects of IL-1β on VLDL-R expression by immunoblot analysis. The downregulation of VLDL-R in response to IL-1β occurred within 12–24 h and was completed in 24–48 h (Fig. 3B). This downregulation by IL-1β occurred for 48 h in a dose-dependent manner (0.3–10 ng/mL; Fig. 3C).


Figure 3
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  		Fig. 3 IL-1β downregulates VLDL-R expression. (A) Cardiac myocytes were treated with 10 ng/mL IL-1β or 1000 U/mL IFN-{gamma} for 48 h and immunostaining for VLDL-R was performed. (B) Cells were treated with 10 ng/mL IL-1β for the indicated periods. (C) Cells were treated for 48 h with IL-1β at the concentrations indicated. Cell lysates were then prepared and analyzed by immunoblotting with antibodies against the VLDL-R or β-actin. Results are expressed as means ± SD (n=3). *p<0.05 and **p<0.01. (D and E) Cells were treated with 10 ng/mL IL-1β for the indicated periods. Total RNA was extracted and analyzed for VLDL-R and iNOS mRNA expression by realtime RT-PCR analysis. Results are expressed as means ± SD (n=3). *p<0.05 and **p<0.01.

 
To investigate whether IL-1β regulates VLDL-R expression at the message level, realtime RT-PCR analysis was performed. Treatment with IL-1β significantly decreased message levels of VLDL-R, but this inhibitory effect was only partial (Fig. 3D). We also evaluated iNOS mRNA expression in the same samples as a positive control, and showed that the iNOS message level was markedly increased by treatment with IL-1β (Fig. 3E).

3.4 IL-1β decreases uptake of DiI-labeled β-VLDL but not DiI-labeled LDL
To explore whether the observed downregulation of VLDL receptor expression by IL-1β was functionally relevant to lipoprotein uptake in cardiac myocytes, the uptake of DiI-labeled lipoproteins was examined. DiI-labeled β-VLDL but not DiI-labeled LDL was incorporated into the cytoplasmic area in unstimulated cardiac myocytes (Fig. 4). Treatment with IL-1β for 48 h markedly inhibited the uptake of DiI-labeled β-VLDL, whereas this treatment had no effect on the uptake of DiI-labeled LDL. We also observed that treatment with IFN-{gamma} had no effect on the uptake of both DiI-labeled β-VLDL and DiI-labeled LDL.


Figure 4
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  		Fig. 4 IL-1β decreases uptakes of β-VLDL but not LDL. Cardiac myocytes were treated with 10 ng/mL IL-1β or 1000 U/mL IFN-{gamma} for 48 h, washed twice, and then treated with 5% LPDS/DMEM containing 10 µg/mL DiI-β-VLDL or DiI-LDL for 3 h. After the cells were washed, the uptake of DiI-β-VLDL or DiI-LDL was observed using fluorescent microscopy. The results are representative of three independent experiments. Magnification, x 100 and x 400.

 
3.5 Inhibition of Hsp90 prevents IL-1β-downregulated VLDL-R expression
To detemine the intracellular signaling pathways involved in IL-1β-downregulated VLDL-R expression, we used tyrosine kinase inhibitors such as HA, GA, and genistein. Pretreatment with HA and GA, but not genistein, prevented the downregulation of VLDL-R expression by IL-1β (Fig. 5A). This inhibitory effect of HA and GA was concentration dependent (Fig. 5B and C). HA and GA have a similar structures and belong to the benzoquinone ansomycin family [21,22]. These 2 agents have been shown to act not only as tyrosine kinase inhibitors, but also as specific inhibitors of cytosolic chaperone Hsp90. Therefore, we tested the effect of another type of Hsp90 inhibitor, radicicol. Pretreatment with radicicol also prevented IL-1β-downregulated VLDL-R expression (Fig. 5D).


Figure 5
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  		Fig. 5 Inhibition of Hsp90 prevents IL-1β-downregulated VLDL-R expression. (A) Cardiac myocytes were pretreated for 1 h with 0.1% DMSO (Cont), 1 µM HA, 1 µg/mL GA, and 10 µM genistein (Geni), and then were treated with 10 ng/mL IL-1β for 48 h. Cell lysates were then prepared and analyzed by immunoblotting with antibodies against the VLDL-R or β-actin. (B–D) Cells were pretreated for 1 h with HA (B), GA (C), and radicicol (D) at the concentrations indicated, and then were treated with 10 ng/mL IL-1β for 48 h. Cell lysates were then prepared and analyzed by immunoblotting with antibodies against the VLDL-R or β-actin. (E) Cardiac myocytes that were transfected with the pSUPERHspiR and pEGFP-C1 were cultured and treated with or without IL-1β for 48 h. The cells were immunostained with antibodies against Hsp90 (red), VLDL-R (red), and GFP (green), stained nuclei with DAPI (blue), and observed by using a confocal microscopy. The cells transfected with siRNA for Hsp86{alpha} (green: arrow) showed marked decrease in the endogenous Hsp90 (red) levels, whereas the cells transfected without siRNA (arrow head) expressed Hsp90 (a and b). The VLDL-R (red) expression was abundantly observed in unstimulated cells (d and e). When cells were stimulated with IL-1β, VLDL-R (red) expression was decreased in untransfected cell (no green), whereas VLDL-R (red) expression was maintained in the cells transfected with siRNA for Hsp86{alpha} (green) (g and h). Bar shows 30 µm. The results are representative of three independent experiments.

 
To test whether the presence of Hsp90 was critical for IL-1β-downregulated VLDL-R expression, we next used a siRNA approach to knock down the endogenous expression of Hsp86{alpha} (mouse/rat homolog for Hsp90). Because cardiac myocytes are one of the difficult cells to transfect, we examined each of the transfected cells under a confocal microscope. When siRNA plasmid for Hsp86{alpha} was cotransfected with GFP (Fig. 5E, a: arrow), the endogenous Hsp90 levels were markedly decreased in the transfected cells but not in the untransfected cells (Fig. 5E, b: arrow). In contrast, the untransfected cells expressed Hsp90 (Fig. 5E, b: arrow head). In unstimulated cells, the VLDL-R expression was abundantly observed (Fig. 5E, e). When cells were stimulated with IL-1β, VLDL-R expression was decreased in untransfected cell (GFP negative cells: Fig. 5E, g and h); however, IL-1β failed to decrease VLDL-R expression in the transfected cells (GFP positive cells: Fig. 5E, g and h). This result suggests that Hsp90 plays a critical role in downregulation of VLDL-R expression by IL-1β in cardiac myocytes.

3.6 No direct association between Hsp90 and VLDL-R
Hsp90 has been shown to prevent degradation of a number of Hsp90 client proteins by its binding. To test the interaction between Hsp90 and VLDL-R in cardiac myocytes, we used co-immunoprecipitation and immunoblotting. Hsp90 from cardiac myocytes was immunoprecipitated using the anti-Hsp90 antibody, and then immunoblotting was performed with anti-VLDL-R antibody. No direct association between Hsp90 and VLDL-R was detected (Fig. 6). As expected, we could detect VLDL-R in the total cell lysates from cardiac myocytes.


Figure 6
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  		Fig. 6 No direct association between Hsp90 and VLDL-R. Cardiac myocytes were stimulated with 10 ng/mL IL-1β for the period indicated (5 m, 5 min; 20 m, 20 min). Immunoprecipitation of the Hsp90 (IP: Hsp90) or VLDL-R (IP: VLDL-R) and immunoblotting with anti-VLDL-R antibody (IB: VLDL-R) or anti-Hsp90 antibody (IB: Hsp90) were carried out as described in the "Materials and methods" section. (A) IP: Hsp90, IB: VLDL-R, (B) IP: VLDL-R, IB: Hsp90. L indicates total cell lysates. No direct association between Hsp90 and VLDL-R was observed. The results are representative of two independent experiments.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
The major findings of this study are: 1) LPS induced alternation of lipid accumulation and VLDL-R expression in the heart; 2) LPS-induced downregulation of VLDL-R was inhibited by the treatment with IL-1RA; 3) IL-1β downregulated VLDL-R expression and attenuated uptake of β-VLDL in cardiac myocytes; 4) Hsp90 was required for IL-1β-downregulated VLDL-R expression. Although it is widely accepted that human septic shock impairs cardiac metabolism and function, the precise mechanism underlying sepsis-induced cardiac alterations is not clear. The findings of this study provide new insight into the pathogenesis of sepsis-induced myocardial dysfunction and lipid metabolism.

During the infection of Gram-negative bacteria, the release of LPS, a component of the outer membrane of bacteria, induces a dysregulated immune response that is characterized by the overproduction of TNF{alpha}, IL-1β, and NO [23,24]. Cardiac metabolism and function are also compromised in the septic state [1–3]. In the present study, we showed that lipid accumulation and VLDL-R expression in the fasting heart was diminished by administration of LPS. Indirect evidence that the VLDL-R may play an important role in cardiac metabolism and function has been reported in several previous studies. Kraemer and his colleagues [25] demonstrated that VLDL-R expression in the myocardium increased progressively with fasting and proposed a potential role for the VLDL-R in the delivery of triglycerides/fatty acids as fuel. Using spontaneously hypertensive stroke-prone rats (SHRs-SP), Masuzaki et al. [26] reported that ventricular VLDL-R mRNA decreased when cardiac hypertrophy was established. The reduced VLDL-R expression in SHRs–SP after cardiac hypertrophy might be linked to the switch in energy substrate from lipid to glucose known to occur in cardiac hypertrophy. Thus, the present study attempted to investigate the role of the VLDL-R and its regulatory mechanisms in the septic heart.

Proinflammatory cytokines such as IL-1β and TNF{alpha} play a pivotal role in sepsis. In fact, serum levels of these proinflammatory cytokines are increased in patients with sepsis [2,23]. A number of experimental and clinical studies have been performed to identify the factors responsible for sepsis and available data support a causative role for TNF{alpha}, IL-1β, and NO [5,6]; however, the factor that mediates sepsis-induced cardiac dysfunction is controversial. In this study, we clearly showed that IL-1β is a key mediator of LPS-induced VLDL-R downregulation. Furthermore, treatment with IL-1β inhibited the uptake of lipoproteins, suggesting that IL-1β induces dysregulation of lipid metabolism of the heart. Supporting this, it has been reported that systemic administration of IL-1β causes dyslipidemia in humans [7]. Although, there is currently no direct evidence that dysregulation of lipid metabolism by IL-1β in the heart induces myocardial dysfunction, we speculate that the effect of IL-1β on lipid metabolism might contribute, at least in part, to myocardial dysfunction in patients with sepsis.

In the present study, although IL-1β stimulation completely downregulated the protein expression of VLDL-R, the substantial levels of mRNA were still observed. We therefore speculated that IL-1β might regulate VLDL-R expression not only through its mRNA expression but also through other mechanism. Supporting this idea, a chaperone molecule Hsp90 is required for IL-1β-mediated VLDL-R downregulation, suggesting that VLDL-R is regulated through both its mRNA and protein levels.

The present study identified Hsp90 as a novel protein modulator of VLDL-R expression. Two major lines of evidence support this conclusion. First, three types of Hsp90 inhibitors (herbimycin A, geldanamycin, and radicicol) reversed the downregulation of VLDL-R expression by IL-1β. Second, the effect of IL-1β on VLDL-R expression was abolished in the Hsp90-downregulated cells. Herbimycin A and geldanamycin belong to the benzoquinone ansamycin family, agents that act as specific inhibitors of both the c-Src family of tyrosine kinases and cytosolic chaperone Hsp90. Because the non-specific tyrosine kinase inhibitor, genistein, could not reverse the downregulation of VLDL-R by IL-1β, we hypothesize that Hsp90 plays a role in this pathway. Supporting this idea, radicicol, another Hsp90 inhibitor also reversed the downregulation of VLDL-R. Hsp90 is an abundant cytosolic protein and binds to a number of protein kinases such as v-Src, raf-1, Bcr–Abl, and ErbB2 [22], thereby maintaining normal enzymatic function as a molecular chaperon. To our knowledge, a substantial role for Hsp90 in lipid metabolism has not been demonstrated. Because Hsp90 generally binds to client proteins, we examined the association between VLDL-R and Hsp90 using an immunoprecipitation technique, but were unable to demonstrate any significant relationship. In addition, Yoshida et al. [27] recently reported that Hsp90 interacts with iNOS protein and enhances its function; however, we could not detect any changes of VLDL-R expression by SNP or L-NMMA in unstimulated and IL-1β-stimulated cardiac myocytes, suggesting that other protein(s) that interact with Hsp90 might mediate the downregulation of VLDL-R expression by IL-1β. Further investigations are required to determine the molecule(s) that might interact with Hsp90 and target VLDL-R in cardiac myocytes.

Under physiological conditions, glucose and fatty acids are the main substrates for energy conversion in the heart [28]. During fasting, the contribution of fatty acids to cardiac energy production increases, whereas the utilization of glucose is reduced [29]. In this regard, Bilsen and his colleagues [30] recently reported that the expression of several molecules involved in cardiac glucose and fatty acid transport and metabolism is altered during fasting. However, the role of VLDL-R in the fasting heart has not been fully understood. In the present study, we found that VLDL-R expression was increased in the fasting heart, suggesting that this fasting-induced VLDL-R expression is one of the responsible mechanisms for adaptation of the cardiac energy metabolism during fasting. We also found that VLDL-R expression was downregulated by administration of LPS. Because fasting is frequently seen in patients with sepsis, the pathological condition such as fasting and sepsis might occur in the clinical setting. Interestingly, lipid accumulation in the heart was not changed by treatment with LPS alone although treatment with LPS alone decreased VLDL-R expression. This finding suggests that other molecules might play a role in the lipid accumulation of the heart without fasting; further investigations are needed to elucidate the precise mechanism of energy metabolism in the heart. Taken together, we speculate that downregulation of VLDL-R in the heart during sepsis may cause a reduction of the cardiac energy substrate supply, resulting in an impairment of cardiac metabolism and function.

In conclusion, the results of this study suggest that IL-1β functions as a principle mediator of changes in cardiac lipid and energy metabolism during sepsis through the downregulation of VLDL-R expression and provides new insight into the essential role of VLDL-R expression in the septic heart.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
We thank Tomoko Hamaji, Junko Yano, and Kazuko Misawa for excellent technical assistance. This study was supported by research grants from the Ministry of Education, Science and Culture, the Ministry of Health, Labor and Welfare (#16590667 to M.T., #17590922 to S.T., and #16390220 to U.I.), and the Daiwa Securities Health Foundation (M.T.).


   Notes
 
Primary review 17 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

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