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Hickson-Bick, Diane L.M.; Jones, Chad; Buja, L. Maximilian

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SHOCK 25(5):p 546-552, May 2006. | DOI: 10.1097/01.shk.0000209549.03463.91
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Sepsis, or gram-negative bacterial endotoxemia, is a life-threatening condition characterized by multiorgan dysfunction and hypotension. Lipopolysaccharide (LPS, endotoxin) is a major structural component of the cell wall of these bacteria and is the mediator for host responses. Exposure of mammalian cells to LPS can lead to profound metabolic responses. These responses include increased production of reactive oxygen species by host inflammatory cells and increased production of arachidonic acid metabolites, including proinflammatory cytokines (1).

Although many local and systemic changes induced by LPS may be the result of activation of inflammatory cells, LPS can elicit direct effects on cardiac myocytes. LPS treatment of adult and neonatal cardiomyocytes in culture induces secretion of tumor necrosis factor alpha (TNF)-α (2). In adult cells, the production of TNF-α has been associated with an increase in cell death by apoptosis (2). In whole animals, cardiac dysfunction induced by LPS exposure is postulated to be a direct result of activation of ventricular myocyte caspase and apoptosis (3). However, treatment of neonatal cardiomyocytes with LPS, at levels far above those necessary to induce TNF-α release, does not cause apoptosis (2). Results from our laboratory and many others have demonstrated a robust apoptotic response of neonatal rat cardiomyocytes in culture to multiple stimuli, including hypoxia (4), saturated fatty acids (5), and doxorubicin (6). However, these neonatal cells exhibit an innate protective phenotype toward cell death induced by the proinflammatory stimulus of LPS. This study was initiated to elucidate these protective pathways in this model system.

LPS treatment of adult and neonatal cardiomyocytes stimulates translocation and activation of the transcription factor nuclear factor (NF)κB (7). NFκB exists in an inactive form in the cytoplasm bound to an inhibitory protein IkappaB (IκB). Phosphorylation of IκB by IκB kinase (IKK) causes dissociation of IκB from NFκB, allowing NFκB to translocate. In adult cardiac cells activation of NFκB translocation is critical for TNF-α production (7). In this report, we demonstrate that NFκB translocation induced by LPS in neonatal rat cardiomyocytes is a result of a degradation of IκBα.

Akt/protein kinase B is a Ser/Thr kinase that can act as a cell survival signaling mediator. Activation by phosphorylation of Akt is via activation of the phosphatidylinositol 3-kinase kinase pathway (8). Akt can phosphorylate and inactivate proapoptotic proteins like caspase-9 and Bad (9, 10). IKK can also be phosphorylated and up-regulated by Akt, leading to an increase in nuclear translocation of NFκB and an inhibition of apoptosis (9, 11). In this study, we examined the role of Akt in protecting neonatal cardiomyocytes against LPS-induced stress.

LPS also induces the activation of enzymes, like cyclooxygenase-2 (COX-2), which can lead to the production of downstream inflammatory molecules in proinflammatory cells (12). The contribution of cyclooxygenase pathways in the response of cardiac myocytes to LPS exposure has not been completely elucidated. We demonstrate that COX-2 activity is activated in the rat neonatal cardiomyocyte in response to LPS exposure, and inhibition of COX-2 in these cells induces apoptosis. However, we also show that increased synthesis of COX-2 is associated with the downstream synthesis of prostaglandins and prostaglandin derivatives, specifically prostaglandins E2 and 15deoxyΔ12-14PGJ2 (15dPGJ2). Inhibition of COX-2 activity can activate caspase-3 activity in these cells, whereas additions of exogenous downstream products of COX-2 activity have varying effects.


Primary cell culture

Neonatal rat ventricular cardiomyocytes were prepared according to McMillin et al.(13) using 1- to 2-day-old Sprague-Dawley pups. Cells were plated at a density of 2 × 106 cells per 60-mm dish and maintained for 48 h in Dulbecco's Modified Eagle's Medium containing 0.3 g/L glutamine, 4.5 g/L glucose, and 10% calf serum before LPS exposure. LPS purified from Escherichia coli K235 (Sigma; St. Louis, MO) was administered to cells in culture (1 μg/mL) in serum containing media without further purification. Wortmannin (Sigma), where appropriate, was added at a concentration of 10 μM 3 h before LPS exposure. NS398 (50 μmol/L; Cayman Chemicals, Ann Arbor, MI) was added 1 h before LPS addition. All experiments conform to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and were approved by the University of Texas Health Science Center in Houston animal welfare committee.

Western blot analysis for Akt, IkBα, Cox-1, and Cox-2

Whole cell lysates were prepared, and proteins were loaded and run on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. Proteins from the gel were transferred to a nitrocellulose membrane electrophoretically. After transfer, the membrane was blocked using gelatin and then treated with rabbit polyclonal antiphospho-Akt (Cell Signaling Technology, Inc., Danvers, MA), anti-IkBα (Santa Cruz Biotechnology Inc., Santa Cruz, CA), goat polyclonal anti-Cox-2, and Cox-1 (Santa Cruz Biotechnology) as primary antibodies. After incubation with the primary antibodies, membranes were washed and incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology). Membranes were washed, and antibody binding was detected using Renaissance Chemiluminescence Reagent Plus kit (NEN, Boston, MA). Signal intensities were quantified by densitometry.

Nuclear extracts and electrophoretic mobility-shift assay

Nuclear extracts were prepared from cultured neonatal rat cardiomyocytes using the method of Muller et al.(14). Phosphatase inhibitors (50 mmol L NaF and 1 mmol L Na3 VO4) were present in all nuclear extraction buffers to preserve phosphorylation states. Annealed oligonucleotides for NFkB (Santa Cruz Biotechnology) were 5end-labeled with {γ32P} adenosine triphosphate by T4 polynucleotide kinase. Extracts were incubated for 20 min at room temperature with 20 μL of reaction buffer that contained 25 mm 4-2-hydroxyethyl-1-piperazineethanesulfonic acid (pH 7.8), 100 mmol L KCl, 2 mmol L dithiothreitol, 0.5% (vol/vol) Nonidet P40, 5% (vol/vol) glycerol, 0.5 mmol L phenylmethanesulfonyl fluoride, 0.2 ng/μL each of aprotinin and leupeptin, and 2.5 ng/μL dIdC. Protein-DNA complexes were run on 4% polyacrylamide gels under nondenaturing conditions in 0.5 × TBE [45 mmol L Tris/borate (pH 8.0) and 1 mmol L EDTA] at 10 V/cm for 2 h. The gels were dried, and Kodak Biomax MS film was exposed to the gels at −70°C overnight and developed. Signal intensities were quantified by densitometry(5). Supershifts were obtained by incubating in the presence of antibody to the P65 subunit of NFκB (Santa Cruz).

TNF-α,PGE2, and 15dPGJ2 immunoassay

After treatment, cardiomyocytes were washed with phosphate-buffered saline and lysed by multiple freeze/thaw cycles. Cell extracts were then used to assay TNF-α levels by enzyme-linked immunoassay (ELISA) (Biosource International, Camarillo, CA) as detailed by the manufacturer. Cell extracts from LPS-treated cardiomyocytes were also used for the detection of PGE2 and 15dPGJ2 production by ELISA (Assay Designs, Inc.) as described by the manufacturer.

Cox-2 activity

This was measured using a colorimetric kit assay (Cayman Chemical) in the presence of a specific Cox-1 blocking antibody under the conditions detailed by the vendor.

Caspase-3 activity assay

Isolated neonatal rat ventricular cardiomyocytes were harvested and lysed in a buffer containing 1% Triton ×100. After lysis, the cells were centrifuged, and the supernatant was collected for assay. Caspase-3 activity, along with the procedure generating positive controls for caspase-3 activation in these cells by incubation with palmitate:BSA, were as previously described by this laboratory(15).

Statistical analysis

Data are represented by mean ± SD. Statistical significance was assessed by one-way analysis of variance. P values ≤ 0.05 were considered significant.


In our cultured neonatal cardiomyocyte system, we observe a rapid increase in nuclear NFκB (Fig. 1A). This is the result of a rapid degradation of IκBα upon exposure to LPS (Fig. 1B). IκBα is bound to NFκB within the cytoplasm. Upon phosphorylation, IκB dissociates from NFκB and is targeted for degradation by ubiquitination (16). Unlike the adult cardiomyocyte (2), this dose of LPS does not induce apoptosis, as assessed by activation of caspase-3 like activity (Fig. 1C). Although changes in NFκB and IκB were observed within minutes of exposure to LPS, changes in apoptotic parameters require a longer time to become apparent. Consequently, caspase-3 activity was measured over a period of many hours. However, exposure on neonatal cardiomyocytes to doses of LPS up to 2 μg/mL for 48 h did not activate caspase-3 activity (data not shown), although the cellular levels of the apoptosis protective protein Bcl-2 fell over this extended period (Fig. 1C, inset).

Fig. 1:
NFκB translocation and caspase-3 activity after LPS exposure. Neonatal rat cardiomyocytes were exposed to LPS at a concentration of 1 μg/mL. A representative electrophoretic mobility-shift assay of NFκB translocation as a function of time after LPS addition is shown in A. IκBα protein degradation in response to LPS was determined by Western blot (B); the inset shows a representative gel. The caspase-3 activity of cells exposed to LPS was also measured (C). The positive control for the caspase-3 activity (Palm) is palmitate:BSA as described in the methods; Inset shows representative Western blot of Bcl-2 levels within these cells All graphs represent the data from at least 3 experiments. Error bars indicates the mean ± SD; *Significantly different from control (no LPS), P ≤ 0.02.

Akt phosphorylation and activation are proposed to initiate protective mechanisms within cardiomyocytes (17). In neonatal rat cardiomyocytes we observed a rapid increase in the level of phosphorylated Akt (pAkt) after LPS treatment (Fig. 2A). However, prior incubation with wortmannin, an inhibitor of PI3 kinase, the enzyme responsible for the phosphorylation of Akt, prevents the activation of Akt (Fig. 2B insert) but does not increase the caspase-3 activity (Fig. 2B).

Fig. 2:
Akt activation after LPS. A, Multiple Western blot analyses of pAkt were quantified and standardized relative to β-Actin; inset includes a representative blot. B, Inhibition of Akt phosphorylation with wortmannin does not increase caspase-3 activity. Cells were preincubated with wortmannin (10 μmol/L) for 2 h then exposed to LPS for 2 h before measurement of caspase-3 activity, a time when Akt activation is close to maximal (see A above). The positive control for the caspase-3 activity is palmitate:BSA as described in the methods; inset is a representative Western blot for pAkt in the presence and absence of wortmannin. n ≥ 3. Values represent the mean ± SD. *Values significantly different from control of no LPS, P ≤ 0.001.

The dose of LPS used in these studies, while not inducing apoptosis in the neonatal cardiomyocytes, is sufficient to stimulate TNF-α production by these cells (Fig. 3). It also induces enzymes associated with the cellular immune response. Cyclooxygenase-2 (COX-2) protein increases in these LPS treated cells, whereas COX-1 expression remains unchanged (Fig. 4).

Fig. 3:
TNF-α production by neonatal rat cardiomyocytes exposed to LPS. Cells in culture were exposed to LPS (1 μg/mL). TNF-α production was measured by ELISA as described in "Materials and methods." Over this time course no increase in TNF-α levels were observed in the absence of LPS. Values indicate the mean ± SD, n ≥ 3; * P ≤ 0.001 vs. no LPS.
Fig. 4:
COX protein expression after LPS. COX-1 and COX-2 protein was detected by Western blot of extracts from neonatal cardiomyocytes exposed to 1 μg/mL LPS over several hours.

Activation of COX-2 leads to the production of downstream inflammatory molecules in other cells. We observed a production of both prostaglandin PGE2 and the cyclopentenone prostaglandin, 15dPGJ2, in neonatal cardiomyocytes exposed to LPS (Fig. 5). 15dPGJ2 attenuates the translocation to the nucleus of NFκB induced by LPS (Fig. 6). This attenuation was not observed with PGE2. Exogenous PGE2 alone did not increase the caspase-3 activity in these cells, whereas exogenous 15dPGJ2 significantly increased caspase-3 activity. However, exogenous 15dPGJ2 and PGE2, when added to neonatal cardiomyocytes in culture in the presence of LPS, can significantly increase the caspase-3 activity in these cells with an additive effect (Fig. 7).

Fig. 5:
PGE2 and 15dPGJ2 in the media of cells exposed to LPS. PGE2 and 15dPGJ2 were assayed by ELISA as described in "Materials and Methods." Values indicate the mean ± SD, n ≥ 3.
Fig. 6:
NFκB translocation in the presence of LPS and 15dPGJ2. Cells were preincubated with varying concentrations of 15dPGJ2 for 2 h then exposed to LPS (1 μg/mL) for 30 min before nuclear extracts are isolated for EMSA. Multiple gels (n = 3) were integrated and are represented graphically. Values indicate the mean ± SD of the measurement; EMSA, electrophoretic mobility shift assay; *P ≤ 0.001 vs. no LPS.
Fig. 7:
Caspase-3 activity in the presence and absence of NS398. Neonatal myocytes were incubated with LPS ± NS398. PGE2 (2 μmol/L), 15dPGJ2(7 μmol/L), and NS398 (50 μmol/L) were added to neonatal cardiomyocytes 1 h before exposure to LPS (1 μg/mL). All cells were exposed to LPS for 20 h before assay of caspase-3 activity. Inset demonstrates the decrease in COX-2 activity when cells were pretreated with NS398 for 1 h followed by LPS exposure for 2 h. All values indicate the mean ± SD, n ≥ 3; *significantly different from cells not exposed to LPS (P ≤ 0.002); **significantly different from cells incubated in the absence of NS398 (P ≤ 0.001); #significantly different from activity measured in the presence of LPS alone (P ≤ 0.001).

Although downstream products of COX-2 activation are capable of inducing apoptosis in neonatal rat cardiomyocytes, specific inhibition of COX-2 using NS398 (Fig. 7 insert) significantly decreased both PGE2 and 15dPGJ2 production while not affecting the production of TNF-α (Table 1). Preincubation with NS398 also significantly induced caspase-3 activation with LPS treatment (Fig. 7). Interestingly, COX-2 inhibition by NS398 in the presence of exogenous 15dPGJ2 or PGE2 alone protected against caspase-3 activation, whereas a combination of these compounds in the presence of LPS and NS398 significantly increased caspase-3 activation to a level observed in the absence of NS398.

Table 1:
NS398 effect on TNFα, PGE2, and 15dPGJ2 production.


One of the profound complications of toxic shock is depression of cardiac function (reviewed in Gates et al.(18). However, the molecular mechanisms involved in this depression are not completely understood. The exact relevance of cell death to the pathogenesis of septic shock is also unclear. Exposure of whole animals, isolated hearts, or cultured adult rat cardiomyocytes to LPS leads to cell death by apoptosis. In our experiments, we demonstrate that neonatal rat cardiomyocytes do not undergo apoptosis when exposed to LPS at levels many times higher than that previously used to induce apoptosis in isolated adult rat cardiomyocytes (2). This absence of apoptosis was surprising in that we did observe a gradual decrease in Bcl-2, a protein that generally plays a protective role against this type of cell death. The induction of apoptosis in the heart can follow several pathways: extrinsic, intrinsic, and possible sarcoplasmic reticulum-driven [for review, see Ref. (19). Activation of caspase-3 is downstream of all of these pathways, and this laboratory has previously shown that activation of this enzyme in neonatal rat cardiomyocytes is associated with DNA laddering and phosphatidylserine translocation, classic cellular markers of apoptosis (5, 15). Caspase-3 activity was therefore used to assess apoptosis in these studies. Our results indicate that the neonatal rat heart has innate protective mechanisms against injury by LPS, which are lost as the heart matures. The nature of these protective mechanisms is unknown, although identification of such mechanisms may represent pharmacological targets to remediate LPS-induced cardiac damage in a clinical setting.

Many cell types, including leukocytes, vascular endothelial cells, smooth muscle cells, cardiomyocytes, and fibroblasts, respond to proinflammatory cytokines by activating NFκB translocation to the nucleus (20-22). Induction of TNF-α production and NFκB translocation by LPS in neonatal mouse cardiomyocytes have previously been reported (7). Blocking NFκB translocation in the mouse also prevented TNF-α production. However, these authors report that the increase in NFκB translocation is a result of increased degradation of cytoplasmic IκBâ, whereas our studies in rat neonatal cardiomyocytes demonstrate an increase in the degradation of IκBα protein. A temporal relationship between nuclear translocation of NFκB and IκBα degradation has been reported in other cell systems (23, 24). Overexpression of a mutant IκBα lacking the wild-type IκBα phosphorylation sites, and thus not subject to inducible degradation, prevents NFκB translocation in rat neonatal cardiomyocytes (25).

Overexpression of cardiac-specific TNF-α in transgenic mice leads to cardiac failure that is a result of increased cardiomyocyte apoptosis (26, 27). TNF-α itself can activate NFκB translocation in neonatal rat cardiomyocytes but does not induce apoptosis (25). Furthermore, Zhang et al.(28) also demonstrated that in the ischemic rabbit heart, NFκB translocation in response to TNF-α is a result of IκBα phosphorylation. NFκB activation also prevents cell death in a number of cell lines(29). In the heart nuclear translocation of NFκB can be protective in some situations, whereas in others, it is detrimental [for review, see Ref. (30). In our neonatal rat cardiomyocyte model, LPS rapidly stimulates NFκB translocation; TNF-α production is also stimulated, but this is temporally much later and not associated with an increase in cardiomyocyte apoptosis.

Akt is a serine-threonine kinase that phoshorylates and inactivates many proteins associated with apoptosis, including Bad and caspase-9 (9, 10). IκK is a substrate for Akt, placing Akt upstream of NFκB in response to some stimuli, including TNF-α (11, 31). In the ischemic rabbit heart NFκB translocation is a result of IκBα phosphorylation by Akt (28). In other reported situations NFκB translocation may initiate activation of Akt (32,33). Our results show that the response to LPS in the neonatal rat cardiomyocyte includes an increase in activated Akt. However, this is downstream of NFκB activation because blocking Akt phosphorylation does not prevent NFκB translocation. Similarly, inhibiting Akt activation does not induce apoptosis after LPS exposure. It should be noted that recent data indicate that the concentration of wortmannin used in these experiments may also be inhibiting other kinases within the neonatal myocyte (34). However, our data indicate that in this model, Akt-regulated pathways are not protecting the neonatal cell from cell death by apoptosis induced by LPS.

The neonatal rat cardiomyocyte demonstrates a protective phenotype against LPS-induced apoptotic cell death. Our data indicate that pathways normally associated with an inflammatory/anti-inflammatory response may change the cell's fate. Prostaglandins, the metabolites of arachidonic acid generated by the cyclooxygenase pathway, also play major roles in cellular responses to inflammation. COX-1 is constitutively active in most tissues, whereas COX-2 is the inducible isoform whose expression is induced by a number of inflammatory stimuli (35). The COX proteins catalyze the conversion of arachidonic acid into PGH2, which can be further metabolized by a variety of enzymes into PGE2, PGI2, PGD2, and thromboxane A2 (36). In the neonatal cardiomyocyte COX-2 protein levels are rapidly increased in response to LPS exposure. Production of both PGE2 and 15dPGJ2 subsequently increase.

The cyclopentenone prostaglandin 15dPGJ2 is a naturally occurring metabolite of PGD2 and may play a role in the resolution of inflammation (37). It is also an endogenous ligand for peroxisome proliferator-activated receptor (PPAR) γ (36, 38). PPARs, when heterodimerized with the retinoid X receptor, act as transcription factors in many cell types, including the heart (39). Many proteins involved in lipid metabolism and metabolic energy supply contain response elements for PPARs. COX-2 is also subject to feedback inhibition by 15dPGJ2 mediated by PPARα (40). We report that addition of pharmacological levels of 15dPGJ2 to neonatal rat cardiomyocytes induces apoptosis, as assessed by caspase-3 activation. Our results in neonatal rat cardiomyocytes indicate that, although 15dPGJ2 and PGE2 can induce apoptosis in these cells in the presence of LPS, the exogenous dose of these metabolites required to stimulate apoptosis is pharmacological rather than physiological; however, this may reflect the very short half-life of these metabolites within the cell (41). Metabolic products of arachidonic acid metabolized by the COX pathway can be potentially apoptotic or antiapoptotic; the balance of these products within a given cell type may define the cellular response to LPS exposure.

The apoptotic effect of 15dPGJ2 has been reported in both polymorphonuclear leukocytes and macrophages (42). Other investigators have reported that inhibition of COX-2 attenuates LPS-induced cardiovascular failure, possibly by reducing the production of inflammatory molecules like PGE2 (43). In the neonatal rat cardiomyocyte inhibition of COX-2 induces apoptosis. These results emphasize the dichotomy observed by other investigators with regard to COX-2 expression after heart injury. In ischemia/reperfusion injury expression of COX-2 has a protective effect (44), whereas after myocardial infarction, an event that increases cardiac COX-2 expression, inhibition of COX-2 activity improves cardiac function (45). Our results are also consistent with a biphasic inflammatory response of the cardiomyocyte to LPS, an early inflammatory response that is resolved later by an anti-inflammatory response. The anti-inflammatory effect of both 15dPGJ2 and COX-2 inhibitors may be related to their ability to induce apoptosis in cardiomyocytes and, in vivo, in infiltrating cells of the immune system. Recent publications also suggest developmental changes in the expression of COX-2 in rats, suggesting that adult cardiomyocytes are resistant to the expression of proinflammatory genes like COX-2 due to an impairment of IKK activation (46). That is not the situation in our neonatal cardiomyocytes. It does suggest that the apoptosis-resistant phenotype in neonates is a function of the ability to mount an inflammatory response. Our results suggest that COX-2 activation results in the production of a number of products and, in the neonatal rat heart, the net balance produces an antiapoptotic phenotype.

In summary, LPS does not induce apoptosis in the neonatal rat cardiomyocyte. NFκB activation occurs rapidly and is not the result of a stimulation of Akt activity. Inflammatory and anti-inflammatory pathways are stimulated in these cells. The neonatal cardiomyocyte represents a cell type in which proapoptotic signaling pathways are attenuated. Apoptosis is a complex process, and the pathways for its induction can vary with stimulus and cell type. Induction of apoptosis can therefore be mediated at many possible sites in multiple pathways. It is probable that the fate of the cardiomyocyte is the result of a combination of a number of mediators. Although beyond the scope of this investigation, a complete proteomic comparison of genes and proteins altered by LPS exposure in neonate and adult could provide a wealth of information about cardiac protective mechanisms. Furthermore, an understanding of the molecular basis of the protection observed in the neonatal cardiomyocyte may identify pharmacological targets for protecting the human heart from sepsis.


This work was supported by the US Army (grant nos. DAMD17-01-2-0047 and DAMD W81XWH-04-02-0035).


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Lipopolysaccharide; septic shock; heart failure; apoptosis; infection/inflammation

©2006The Shock Society