Lipopolysaccharide Internalization Activates Endotoxin-Dependent Signal Transduction in Cardiomyocytes Douglas B. Cowan, Sabrena Noria, Christof Stamm, Lina M. Garcia, Dimitrios N. Poutias, Pedro J. del Nido, Francis X. McGowan, Jr Abstract—We tested the hypothesis that bacterial lipopolysaccharide (LPS) must be internalized to facilitate endotoxindependent signal activation in cardiac myocytes. Fluorescently labeled LPS was used to treat primary cardiomyocyte cultures, perfused heart preparations, and the RAW264.7 macrophage cell line. Using confocal microscopy and spectrofluorometry, we found that LPS was rapidly internalized in cardiomyocyte cultures and Langendorff-perfused hearts. Although LPS uptake was also observed in macrophages, only a fraction of these cells were found to internalize endotoxin to the extent seen in cardiomyocytes. Colocalization experiments with organelle or structure-specific fluorophores showed that LPS was concentrated in the Golgi apparatus, lysosomes, and sarcomeres. Similar intracellular localization was demonstrated in cardiomyocytes by transmission electron microscopy using gold-labeled LPS. The internalization of LPS was dependent on endosomal trafficking, because an inhibitor of microfilament reorganization prevented uptake in both cardiomyocytes and whole hearts. Inhibition of endocytosis specifically restricted early activation of extracellular signal–regulated kinase proteins and nuclear factor-B as well as later tumor necrosis factor-␣ production and inducible nitric oxide synthase expression. In conclusion, we have demonstrated that bacterial endotoxin is internalized and transported to specific intracellular sites in heart cells and that these events are obligatory for activation of LPS-dependent signal transduction. (Circ Res. 2001;88:491-498.) Key Words: Golgi apparatus 䡲 microfilaments 䡲 endocytosis 䡲 lysosomes 䡲 signal transduction
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ndotoxin or lipopolysaccharide (LPS) from the outer membrane of Gram-negative bacteria is the primary trigger of the systemic inflammatory response in sepsis.1 Sepsis or septic shock develops in ⬎500 000 patients each year in the United States alone, of which nearly half die.2 The clinical manifestations of sepsis are in large part attributable to LPS-induced signal transduction and gene expression in both myeloid cells (eg, macrophages) and nonmyeloid cells (eg, endothelial cells and cardiomyocytes). These events are the primary cause of myocardial dysfunction in sepsis, which is an important determinant of patient outcome. In the heart, substantial evidence has been collected that indicates LPS exerts its effects in 2 overlapping phases. Reduced systolic function and contractile reserve occur within minutes to hours of LPS exposure.3 These phenomena occur in the absence of systemic acidosis, hypotension, or decreased coronary perfusion. Early myocardial dysfunction has been related to direct LPS effects and rapid LPSstimulated production of proinflammatory cytokines, such as tumor necrosis factor-␣ (TNF-␣).4,5 Additional elaboration of proinflammatory cytokines and other mediators in response
to the LPS signal results in injury from a variety of mechanisms that include free radical production, nitric oxide generation, and arachidonic acid metabolite release. These events result in progressive contractile dysfunction, diminished -adrenergic responsiveness, impaired oxidative metabolism, and cell death. Therefore, defining how the LPS signal is transduced in the heart is relevant to understanding the pathophysiology of myocardial dysfunction during sepsis. The delayed effects of LPS in the heart have been well studied; however, little is known about the process of early LPS signaling or injury. Our laboratory has previously shown that LPS rapidly activates members of the extracellular signal–regulated kinase (ERK), signal transducer and activator of transcription (STAT), and nuclear factor-B (NF-B) signal transduction cascades in cardiomyocytes.6 Unlike cells of reticuloendothelial origin, signaling through these pathways seems to be receptor-mediated but independent of the glycosyl phosphatidylinositol-linked receptor CD14 and lipopolysaccharide-binding protein.6 The signaling events that precede activation of these pathways in cardiac cells, however, have not been thoroughly studied.
Original received October 3, 2000; revision received December 22, 2000; accepted January 11, 2001. From the Departments of Anesthesia (D.B.C., D.N.P., F.X.M.) and Cardiac Surgery (L.M.G., C.S., P.J.D.), Children’s Hospital and Harvard Medical School, Boston, Mass, and the Department of Laboratory Medicine and Pathobiology (S.N.), University of Toronto, Toronto, Ontario, Canada. Correspondence to Douglas B. Cowan, PhD, Department of Anesthesia, Enders Room 1355, Children’s Hospital, 300 Longwood Ave, Boston, MA 02115. E-mail
[email protected] © 2001 American Heart Association, Inc. Circulation Research is available at http://www.circresaha.org
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Recently, a number of laboratories have shown that LPS is internalized by several cell types,7–11 but the role of LPS uptake in activating signal transduction remains controversial.12–15 Polymorphonuclear leukocytes and HeLa cells endocytically transport monomeric LPS to the Golgi apparatus.8 Although CD14 may be important in monomeric LPS recognition and transfer to either a coreceptor or directly into the plasma membrane in myeloid cells, the intracellular trafficking of LPS to the Golgi appears independent of this receptor.11 On the other hand, LPS aggregates are likely transported in macrophages to lysosomes in conjunction with CD14, where acyloxyacyl hydrolase deacylates LPS.7,10,11,16 The latter function is ostensibly more relevant to the detoxification and clearance of endotoxin rather than the initiation of signaling.11,17 LPS-mediated signal transduction is presently believed to be governed by the TLR4 transmembrane receptor in both cardiac and myeloid cells.1,18 –22 Because endocytosis of ligand-activated cell-surface receptors often regulates signal transduction and gene expression,23 we have studied the relationship between LPS intracellular trafficking and signal activation in the heart. Given the complexity and diversity of later LPS-induced effects in this organ, it is evident that identifying the early mechanisms of endotoxin signaling in the myocardium is essential for developing novel and specific strategies to prevent cardiac dysfunction during sepsis. In the present study, we found that LPS was rapidly internalized in both cardiomyocytes and perfused whole hearts. In cardiomyocytes, LPS was sorted through an endosomal pathway to the Golgi complex, lysosomes, and contractile apparatus. This process was linked with the immediate activation of ERK and NF-B signaling pathways and the later production of TNF-␣ and nitric oxide. Our findings indicate that endocytic membrane trafficking and retrograde transport of LPS regulates endotoxin-dependent signal transduction in cardiac muscle.
production in cardiomyocytes and RAW264.7 cells to the same extent as unlabeled LPS.
Treatments and Staining Cells were treated with 0.01 to 1 g/mL labeled LPS. Some cells were treated with 50 nmol/L LysoTracker Red DND-99, 50 nmol/L BODIPY TR C5-ceramide, or 500 nmol/L MitoTracker CMX Ros (Molecular Probes) along with BODIPY FL–labeled LPS (BODIPY FL-LPS). Others were pretreated for 30 minutes with 1, 10, or 100 mol/L cytochalasin D (Sigma) before LPS or 50 and 500 mol/L H2O2 treatment in combination with cytochalasin. For staining, treated cultures were fixed and mounted directly or stained with either TRITC-phalloidin (Sigma) or Texas Red X-phalloidin (Molecular Probes). Perfused hearts were treated with Texas Red X-LPS⫾10 mol/L cytochalasin D and then fixed, paraffinembedded, sectioned, and mounted for visualization.
Confocal Laser Microscopy Slides were visualized using a BioRad MRC1024 confocal microscope with a Nikon ⫻60 oil immersion objective, NA⫽160/0.17. BODIPY FL was excited at 488 nm and detected between 506 to 538 nm. TRITC and Texas Red X were excited at 568 nm and detected between 589 and 621 nm. Optical sections (0.5 m) were merged and projected with the BioRad software.
Transmission Electron Microscopy LPS-Au–treated cells were fixed in 2.5% grade I glutaraldehyde (Sigma) and silver (Ag)-enhanced (Nanoprobes) before staining with 0.25% uranyl acetate and 0.5% osmium tetroxide (Electron Microscopy Sciences). Sections (60 nm) were cut with a Reichert Ultracut-S ultramicrotome and mounted on copper grids (200 mesh) for electron microscopy.
Immunoblot Analyses Immunoblotting was performed as described earlier.6 The anti-ERK1 (K-23) antibody (Santa Cruz) was used at 0.1 g/mL to detect ERK1/2, whereas the antiphospho-p44/42 mitogen-activated protein kinase (MAPK) E10 antibody (New England Biolabs) was diluted to 0.05 g/mL to detect phosphorylated (active) forms of ERK1/ERK2. The anti-inducible nitric oxide synthase (iNOS; NOS type II) antibody (Transduction Laboratories) was diluted to 0.1 g/mL.
Electrophoretic Mobility Shift Assays
Materials and Methods Cell Culture and Isolated Perfused Heart Preparations Animal procedures received institutional approval and were conducted according to National Institutes of Health guidelines (publication No. 85-23, 1985). Wistar rat (Charles River Laboratories, Portage, Manitoba, Canada) cardiomyocytes and the RAW264.7 mouse macrophage cell line (American Type Culture Collection) were cultured as described earlier.6 Adult rat hearts were Langendorff-perfused with Krebs-Henseleit buffer at 10 mL/min constant flow essentially as described.3 A spectrofluorometer (SLMAminco) measured real-time emission light from hearts at 550 to 650 nm after excitation at 524 nm.24
Fluorescent and Gold Labeling of LPS Salmonella typhosa LPS (Sigma) was labeled with BODIPY FL (4,4-difluoro-4-bora-3a, 4a-diaza-s-indacene fluorescein), Oregon Green 488, or Texas Red X succinimidyl ester derivatives (Molecular Probes), as described earlier.25 LPS was also labeled with 1.4-nm-diameter mono-Sulfo-N-hydroxysuccinimide ester Nanogold particles (Nanoprobes) and purified by gel filtration. The labeling efficiency for each of the succinimidyl ester compounds was calculated to be 82.6% to 93.5% on the basis of absorbance measurements and known extinction coefficients. In addition, both fluorophore and gold-labeled LPS (LPS-Au) stimulated TNF-␣ secretion and nitrite
Nuclear extracts were isolated from LPS-treated cardiomyocytes using the method of Andrews and Faller,26 and gel shift assay reactions were carried out as described previously.6
Measurement of TNF-␣ and Nitrite Levels
Rat TNF-␣ levels were determined in culture media samples using the Quantikine (R&D Systems) sandwich ELISA. Nitric oxide production was determined in culture media using Greiss reagent.27 Comparisons of TNF-␣ and nitrite production were made using an ANOVA followed by the Tukey-Kramer test.
Results LPS Is Internalized in Cardiomyocyte Cultures and Whole Hearts To determine whether BODIPY FL-LPS was internalized in cardiomyocyte cultures, we incubated cells with 1 g/mL labeled LPS for various times (Figures 1A through 1D). Fixed cardiomyocytes were stained for filamentous actin (F-actin) (red). A diffuse distribution of LPS (green) was apparent in cardiomyocytes by 30 minutes, with more intense globular staining at perinuclear locations at 60 minutes and 24 hours. Labeled LPS was also observed to localize to a region consistent with either the A- or H-band of the sarcomere.
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Figure 1. Confocal photomicrographs of cardiomyocytes exposed to BODIPY FL-LPS for various times. Cells were incubated with 1 g/mL fluorescently labeled S. typhosa LPS (green) for 0 (A), 30 (B), and 60 minutes (C) and 24 hours (D). After fixation, cells were cross-stained for F-actin with Texas Red X-phalloidin (red) and examined. The micrographs represent a series projection of 0.5-m optical sections from the basal to the apical surface of the cells. Scale bars⫽10 m. Experiments were repeated 8 times.
Serial 0.5-m mid (Z)-plane optical sections of cardiomyocytes treated with labeled LPS for 60 minutes are shown in Figure 2 (top). An intense punctate staining pattern was observed in the perinuclear region in addition to staining in the contractile apparatus. For comparison, BODIPY FL-LPS staining of RAW264.7 macrophages indicated that ⬇15% of the macrophages avidly internalized LPS whereas the remaining cells exhibited relatively low levels of uptake (bottom). Despite variability in the degree of internalization, all macrophages were found to contain labeled LPS. To establish that LPS internalization was not attributable to the BODIPY FL compound, we labeled endotoxin with 2 structurally different fluorophores. LPS labeled with either Oregon Green 488 or Texas Red X succinimidyl esters exhibited a similar pattern of staining as demonstrated for BODIPY FL-LPS in both cardiac myocytes and RAW264.7 cells (not shown). Furthermore, unreacted BODIPY FL isolated from the gel filtration column used to purify labeled LPS exhibited no internalization. Similar levels of internalization were seen in both cell types at LPS concentrations ranging from 0.1 to 10 g/mL; however, detection of fluorescent LPS at concentrations ⱕ0.01 g/mL was more difficult than that observed at higher concentrations. Langendorff-perfused whole hearts were also found to internalize LPS. Perfused hearts were fixed and sectioned to confirm the intracellular localization of Texas Red X-LPS (Figures 3A through 3C). Figure 3A shows the level of autofluorescence and background fluorescence in a control heart perfused with unreacted fluorophore alone, whereas
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Figure 2. Photomicrographs of cardiomyocyte cultures (CMCs) and RAW264.7 macrophages treated with 1 g/mL BODIPY FL-LPS for 60 minutes. Cells were stained for F-actin and examined on a confocal microscope. LPS appears green and F-actin appears red. A through D, 0.5-m (top) or 1-m (bottom) optical sections midway between the apical and basal surfaces of the cells and the projected series of these sections (E). Scale bars⫽50 m. Experiments were repeated 6 times.
Figures 3B and 3C demonstrate endotoxin uptake in hearts perfused with Texas Red X-LPS. Figure 3D shows the rate of Texas Red X-LPS uptake in a working heart model. Texas Red X-LPS uptake occurred within 10 minutes. To demonstrate that LPS was localized to an intracellular space, we perfused hearts for 8.5 minutes with labeled LPS followed by a washout period using perfusate alone (Figure 3E). A drop in fluorescent emission was seen between 8.5 and 10 minutes, indicating that labeled LPS was eliminated from the vascular lumen. After the initial decline in signal intensity, cardiac fluorescent emission stabilized (10 to 30 minutes), demonstrating that Texas Red X-LPS remained within heart cells.
LPS Is Transported to the Golgi Complex and Lysosomes The intracellular location of LPS was investigated using 2 strategies. In the first, cardiomyocyte cultures were treated with 0.1 g/mL BODIPY FL-LPS for 60 minutes. Simultaneously, the cells were exposed to 3 different organellespecific stains (BODIPY TR C5-ceramide, Golgi apparatus; LysoTracker Red DND-99, lysosomes; and MitoTracker CMX Ros, mitochondria) and then visualized on a confocal microscope. The second approach used an electron microscope and LPS labeled with 1.4-nm-diameter gold particles. Figure 4 shows typical results from the experiments using confocal microscopy. For simplicity, cardiomyocytes with a single nuclei are depicted; however, binucleated cells exhibited comparable staining patterns. The left column of the photomicrographs (panels A, D, and G) shows the perinuclear
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Figure 3. Photomicrographs of rat heart tissue perfused with Texas Red X-LPS for 30 minutes. Hearts were either perfused in the absence (A) or presence (B and C) of 1 g/mL fluorescently labeled LPS and examined by confocal microscopy. Micrographs represent 0.5-m optical sections of tissue aligned in either the longitudinal (A and B) or transverse (C) orientation. Arrowheads indicate blood vessels. Scale bars⫽50 m. D and E, Spectrofluorometric measurement of Texas Red X-LPS uptake in the left ventricle of perfused hearts. Fluorescence at 614 nm in hearts either continuously perfused with LPS suspended in Krebs-Henseleit buffer for 30 minutes (D) or perfused for 8.5 minutes with LPS followed by 21.5 minutes perfusion with buffer alone (E). Values are expressed as a percentage of the peak fluorescence and plotted as mean⫾SEM (n⫽4). All measurements have been adjusted for background autofluorescence and intraluminal vascular fluorescence.
staining by LPS, as noted in Figures 1 and 2. The center column (panels B, E, and H) shows the organelle-specific stains for the Golgi complex (top), lysosomes (middle), and mitochondria (bottom). The right column (panels C, F, and I) reveals the merged images from the corresponding left and center columns. Any overlap in the areas stained with LPS (shown in green) and the organelle-specific stains (shown in red) appear as yellow in the right column. As demonstrated in panels C and F, there is considerable overlap between LPS and Golgi or lysosome-specific stains. The yellow staining seen in panel I is bleed-through between red and green fluorescent channels and is not considered colocalization. This assertion was confirmed when individual 0.5-m optical sections were examined rather than images of the projected series shown in Figure 4 (not shown). The greatest degree of LPS colocalization was seen with the Golgi-specific stain,
Figure 4. Colocalization of BODIPY FL-LPS with Golgi-, lysosomal-, or mitochondrial-specific fluorophores. Primary cardiomyocytes were treated with 0.1 g/mL fluorescently labeled LPS and BODIPY TR C5-ceramide (A through C), LysoTracker Red DND-99 (D through F), or MitoTracker CMX Ros (G through I) for 60 minutes. BODIPY FL-LPS is shown in green (A, D, and G), and C5 ceramide (B), LysoTracker (E), or MitoTracker (H) is shown in red. Merged images of A and B, D and E, and G and H are depicted in C, F, and I, respectively. Colocalization of red and green pixels appears in yellow. A projection of 0.5-m optical sections is shown. Scale bars⫽10 m. Experiments were repeated 6 times.
with nearly all of the spherical bodies observed in panel C appearing yellow. The merged image in panel F shows that a majority of the lysosomes colocalize with LPS; however, individual green and red areas of staining are also apparent (particularly when discrete optical sections were examined), indicative of a less-uniform distribution of LPS in the lysosomes compared with that of the Golgi. For transmission electron microscopy, LPS was labeled with NanoGold particles rather than a gold colloid to minimize the size of the attached label and ensure uniformity of particle size. LPS-Au-Ag associated with structures, consistent with the interior of endosomes or early lysosomes (Figures 5A through 5C). In most cases, LPS-Au-Ag was associated with membranous regions within these vesicles, similar to the findings of Kreigsmann et al.28 It was also observed that some LPS-Au-Ag was associated with the plasma membrane (Figure 5A); however, surface staining was never seen at sites consistent with caveolae or clathrincoated pits. A longer treatment (60 minutes) resulted in localization of LPS-Au-Ag in lysosomes (arrows in Figures 5D and E) or compact vesicular structures surrounding the Golgi complex (Figure 5F). These vesicles were primarily located near trans-face and alongside the Golgi. Labeled LPS was not found associated with the cisternae of the Golgi despite the high level of colocalization demonstrated in Figure 4.
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Figure 5. Transmission electron microscopy of cardiomyocytes treated with 0.01 g/mL LPS-Au. NanoGold-labeled LPS was applied to cell cultures for 5 minutes (A through C) or 60 minutes (D through F) before fixation and processing. N indicates nuclei. Arrows point toward endosomes (A through C), lysosomes (D and E), and vesicles surrounding the Golgi (F). Other areas labeled with LPS-Au-Ag include the cell surface (A), a residual body (below the arrow in B), and several lysosomes (C and D). Areas not found to contain LPS-Au include the nucleus, mitochondria, lipid droplets, and endoplasmic reticulum. Scale bars⫽200 nm. Experiments were repeated 4 times.
Activation of Signal Transduction by Lipopolysaccharide Is Dependent on Uptake To ascertain whether LPS internalization and intracellular trafficking are required for activation of signal transduction in cardiomyocytes, we inhibited endocytosis with cytochalasin D.29,30 Figure 6 demonstrates that in perfused whole hearts, 10 mol/L cytochalasin D completely blocked the internalization of Texas Red X-LPS. Similarly, BODIPY FL-LPS was not internalized in cardiomyocyte cultures treated with cytochalasin D (not shown). We investigated whether cytochalasin D blocked early (ⱕ60 minutes), intermediate (4 hours), or late (24 hours) signaling in cardiomyocytes. Figure 7A shows the activity of ERK proteins at 10 minutes after LPS or H2O2 treatment in the presence of 1, 10, and 100 mol/L cytochalasin D. ERK was maximally phosphorylated 10 minutes after LPS expo-
sure, as shown in lanes 1 (untreated) and 2 (0.1 g/mL LPS), in agreement with our previous observations.6 Increasing doses of cytochalasin D (lanes 3 through 5) attenuated ERK phosphorylation, whereas cytochalasin D alone had no effect on ERK activity (not shown). To demonstrate that cytochalasin was inhibiting ERK phosphorylation specifically through prevention of endocytosis and not via inhibition of the MAPK pathway, 50 mol/L hydrogen peroxide (H2O2) plus cytochalasin D was used to treat cardiomyocytes. ERK proteins were activated by H2O2 in the presence of 10 mol/L cytochalasin D (lane 6). Moreover, the ability of nuclear proteins to specifically bind a NF-B consensus binding sequence after 60 minutes of LPS treatment (Figure 7B; compare lanes 1 and 2) was reduced in the presence of escalating concentrations of cytochalasin (lanes 3 through 5). Inhibitor alone had no effect
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Figure 6. Texas Red X-LPS uptake in hearts perfused in the presence ( e ) or absence (䉫) of an inhibitor of endosomal trafficking. Cytochalasin D (10 mol/L) limited internalization of 1 g/mL fluorescently labeled LPS to baseline levels, as measured by fluorescence at 614 nm. Values are expressed as fold increase in peak fluorescence and plotted as mean⫾SEM (n⫽4). All measurements have been adjusted for background autofluorescence and intraluminal vascular fluorescence of the labeled LPS.
on NF-B binding (lane 6). The specificity of the DNA protein interaction was established in lanes 7 and 8. A 50-fold molar excess of identical unlabeled binding site abolished the shifted complex (lane 7), whereas an unrelated but similarly sized sequence had no effect on complex formation at a 500-fold molar excess (lane 8). The composition of the NF-B complex was investigated by preincubating nuclear extracts with antibodies directed against either NF-B p50 or p65 subunits (lanes 9 and 10, respectively). Only the anti-p65 antibody efficiently supershifted the complex, indicating that either the p50 antibody did not bind to its target antigen well or that p50 was not a part of the NF-B complex. The latter possibility would indicate that other NF-B subunits (ie, p52) might be involved in orchestrating LPS-induced gene expression. In addition, 500 mol/L H2O2 was found to cause specific binding of nuclear proteins to the NF-B consensusbinding sequence in the presence of 10 mol/L cytochalasin D (not shown). In Figure 8A, the effect of inhibiting LPS uptake on TNF-␣ production from cardiomyocyte cultures treated for 4 hours was established. Concentrations of cytochalasin D (1, 10, and 100 mol/L) decrease LPS-induced TNF-␣ production. This effect was the result of inhibiting LPS internalization and did not result from preventing TNF-␣ secretion, because there was no detectable accumulation of intracellular TNF-␣ in cell lysates from these cultures (not shown). Untreated cells and cultures treated with only cytochalasin D were not significantly different (lanes 1 and 6). The expression and activity of iNOS 24 hours after 0.1 g/mL LPS administration in the presence of 0, 1, 10, and 100 mol/L cytochalasin D is shown in Figures 8B and 8C (lanes 2 through 5).
Discussion We have provided evidence that LPS is internalized in both cardiomyocyte cultures and the cells of Langendorff-perfused hearts. In addition, intracellular LPS was localized to the cardiomyocyte Golgi complex, lysosomes, and contractile apparatus. It is likely that the intracellular transport of LPS depends on its molecular composition at the plasma membrane. In other cell types, it has been shown recently that monomeric LPS is transported to the Golgi apparatus,
Figure 7. Top, Immunoblot analysis of phosphorylated and total ERK1/2 in cardiomyocytes treated with BODIPY FL-LPS and cytochalasin D (CD). Cells were pretreated for 30 minutes with 1 (lane 3), 10 (lanes 4 and 6), and 100 (lane 5) mol/L cytochalasin D before being exposed to 0.1 g/mL labeled LPS (lanes 2 through 5) or 50 mol/L H2O2 (lane 6) for 10 minutes in the presence (lanes 3 through 6) or absence (lane 2) of cytochalasin. Untreated cell lysates are shown in lane 1. Bottom, Electrophoretic mobility shift assay using a NF-B consensus-binding site reacted with LPS-treated and cytochalasin D–treated cardiomyocyte nuclear extracts. Extracts (lanes 1 through 5) were derived from cells treated for 60 minutes as described for the respective lanes above. Extract from cells treated with 100 mol/L cytochalasin D alone is shown in lane 6. Lanes 7 through 10 contained extracts from cardiomyocytes treated with 0.1 g/mL LPS. In these reactions, lane 7 contained unlabeled NF-B at a 50-fold molar excess and lane 8 had unlabeled AP-1– consensus binding site at a 500-fold molar excess. Lanes 9 and 10 contained 0.5 g anti–NF-B p50 and p65 supershift antibodies. Specifically bound DNA protein complexes and free probe are indicated. Experiments were repeated 5 times.
whereas aggregates move into lysosomal compartments.8,11 Although LPS in solution would presumably exist as an aggregate, we found endotoxin in both the Golgi complex and lysosomes (Figures 5 and 6), indicating that LPS may be internalized in both monomeric and aggregate form in cardiomyocytes. Selective intracellular sorting of endotoxin may depend on molecular weight or conformation, because labeling compounds that vary greatly in size, to a certain extent, affected the intracellular localization of LPS. Fluorescently labeled LPS was found largely in the Golgi complex with less in the lysosomal compartments (Figure 4). LPS-Au, on the other hand, was concentrated in small vesicles surrounding, but not within, the cisternae of the Golgi, in addition to being within lysosomes and endosomes (Figure 5). The large size of the NanoGold conjugate (15 000 Mr) may cause LPS to be trafficked within the cardiomyocyte in a manner typical of endotoxin aggregates. By comparison, BODIPY FL (⬎500 Mr) may sort in a manner more representative of LPS monomers.
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Figure 8. A, TNF-␣ production in cardiomyocytes treated with BODIPY FL-LPS and cytochalasin D (CD). Cells were pretreated for 30 minutes with 1, 10, and 100 mol/L cytochalasin D (bars 3 through 5, respectively) before being exposed to 0.1 g/mL labeled LPS for 4 hours (bars 2 through 5) in the presence (bars 3 through 5) or absence (bar 2) of cytochalasin. After treatments, culture media were analyzed for TNF-␣, and untreated cardiomyocyte culture media were used as a negative control (bar 1). Results are presented as pg/mL TNF-␣ (mean⫾SD), where n⫽4 and all experiments had an equal number of cells. There was a significant increase (*P⬍0.001) in TNF-␣ secretion in cells treated with LPS alone (bar 2) compared with untreated cardiomyocytes (bar 1), whereas increasing concentrations of cytochalasin D (bars 3 through 5) significantly reduced (**P⬍0.001) TNF-␣ production. Cytochalasin D alone (bar 6) did not significantly alter TNF-␣ secretion compared with the control (bar 1). B, Immunoblot analysis of iNOS. C, Nitrite production in cardiomyocyte cultures treated with BODIPY FL-LPS and cytochalasin for 24 hours. Samples were run in the same order (ie, 1 to 6) as described for TNF-␣. A significant increase (*P⬍0.001) was demonstrated in nitrite accumulation in cells treated with LPS alone (bar 2) compared with untreated cardiomyocytes (bar 1). Increasing concentrations of cytochalasin D (bars 3 through 5) significantly reduced (**P⬍0.001) nitric oxide production. Results are presented as mol/L nitrite (mean⫾SD), where n⫽4 and values have been adjusted for total cardiomyocyte protein.
We also found LPS associates with the contractile apparatus. The lack of overlap between the phalloidin and BODIPY FL-LPS staining suggests that LPS associates with the H-band of sarcomeric thick filaments, which are composed principally of myosin. It is possible that some of the early cardiac contractile effects of LPS may be attributed the accumulation of endotoxin in the sarcomere (Figures 1 and 2).3 In addition, because endotoxin was found to persist in the Golgi apparatus for at least 24 hours (Figure 1 and data not shown), LPS has the potential to obstruct Golgi and endoplasmic reticular processing of cellular proteins destined for membrane compartments or the extracellular space. Because
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LPS cannot be metabolized in the Golgi complex, a situation reminiscent of the endoplasmic reticulum (ER)-overload response may be occurring.8,31 In the ER-stress response, the NF-B signaling pathway is activated because of an accumulation of proteins in the ER. This could occur because of a backlog from the Golgi complex as the result of LPS deposition in that organelle. Our present and earlier6 results are consistent with this phenomenon but do not exclude other possibilities. LPS was also internalized in the cardiomyocytes and vascular cells of perfused whole hearts (Figure 3). This finding represents the first demonstration of LPS internalization in a solid organ. The spectrofluorometer used for some of these studies measured output signal from a section of the left ventricular free wall. The effective excitation light penetration from the 400-W xenon lamp used in these experiments was ⬇4 mm, and the resultant emission light was estimated to be unaffected by tissue absorbance.24 Microscopic examination of tissue sections from the perfused hearts verified that fluorescent LPS was evenly distributed intracellularly throughout the ventricular wall. A longer wavelength fluorophore (Texas Red X) was used to avoid the large amount of autofluorescence attributable to myoglobin that is observed near the peak emission wavelength for BODIPY FL or Oregon Green 488. We observed that uptake of LPS can be completely prevented by treating the perfused hearts with cytochalasin D just before and during the administration of Texas Red X-LPS (Figure 6). There are several studies in the literature that support our finding that LPS internalization is dependent on microfilament reorganization.12,13 Although cytochalasins have been used to block LPS uptake in several cell types, this molecule has also been shown to prevent downstream signaling as a consequence of LPS exposure.32–34 Interestingly, Poussin et al12 showed that cytochalasin D did not prevent LPS-dependent p38 MAPK and NF-B activation in THP-1 cells. In their study, cytochalasin D actually increased interleukin-8 secretion after LPS treatment.12 Additionally, we have demonstrated that cytochalasin D treatment of cardiomyocytes, in a dose-dependent manner, prevented the immediate activation of ERK and NF-B signaling pathways (Figure 7) and the delayed production of TNF-␣ and nitric oxide (Figure 8). Activation of these signaling proteins was specifically attributable to the prevention of internalization of LPS and not the result of direct inhibition of the ERK and NF-B pathways, because H2O2 could stimulate these signaling cascades in the presence of cytochalasin. Hydrogen peroxide has previously been proven to stimulate both of these pathways in cardiomyocytes,35,36 likely through a receptor-independent means. On the basis of the assumption that in cardiac muscle cells TNF-␣ secretion leads to inducible nitric oxide synthase gene expression,37 it is not surprising that microfilament disruption prevents nitric oxide production, because TNF-␣ is regulated by the NF-B and ERK pathways.6 Consequently, neither pathway can be activated in response to LPS, when internalization is blocked (Figure 7). There is presently insufficient evidence to conclude that LPS signaling occurs as a direct result of concentration in the
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Golgi apparatus or other cellular compartments. On the basis of the rapidity of some responses, it is probable that signals are generated from an intermediate structure like the endosome. This supposition is supported by the observation that although LPS-Au was biologically active (as measured by the ability to stimulate TNF-␣ secretion and nitric oxide production), it was not localized within the Golgi complex. Whether LPS is receptor associated (eg, TLR4) within cellular compartments or integrated into an intracellular membrane also remains to be elucidated. Internalization of ligand-activated receptors is a well-recognized means of modulating signal transduction, with most examples of signaling from within endosomes indicating that ligand receptor complexes are associated with caveolae or clathrin-coated pits.23 We did not observe LPS associated with either type of structure (Figure 5), suggesting that other mechanisms may be involved. In conclusion, we have shown that LPS is internalized and sorted to specific locations in cardiomyocytes and that these events are required for endotoxin-dependent signal activation. A complete understanding of the initial events that result in production of deleterious gene products or directly interfere with contractile function may lead to the development of therapies designed to protect the heart from endotoxin exposure.
Acknowledgments This work was supported by the Charles H. Hood Foundation of Boston, the National Institutes of Health (grants HL52589 and HL46207), and the Children’s Hospital Anesthesia Foundation.
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Lipopolysaccharide Internalization Activates Endotoxin-Dependent Signal Transduction in Cardiomyocytes Douglas B. Cowan, Sabrena Noria, Christof Stamm, Lina M. Garcia, Dimitrios N. Poutias, Pedro J. del Nido and Francis X. McGowan, Jr Circ Res. 2001;88:491-498 doi: 10.1161/01.RES.88.5.491 Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2001 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. Online ISSN: 1524-4571
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