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Am J Physiol Heart Circ Physiol 294: H1490-H1496, 2008. First published January 25, 2008; doi:10.1152/ajpheart.00910.2007
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Negative inotropic effects of high-mobility group box 1 protein in isolated contracting cardiac myocytes

Huei-Ping Tzeng,1,2,* Jinping Fan,1,2,* Jesus G. Vallejo,1,3 Jian Wen Dong,1,2 Xiongwen Chen,4 Steven R. Houser,4 and Douglas L. Mann1,2

1Department of Medicine, Winters Center for Heart Failure Research; 2Texas Heart Institute at Saint Luke's Episcopal Hospital; 3Department of Pediatrics, Section of Infectious Diseases, Baylor College of Medicine and Texas Children's Hospital, Houston, Texas; 4Molecular and Cellular Cardiology Laboratories, Cardiovascular Research Group, Temple University School of Medicine, Philadelphia, Pennsylvania

Submitted 6 August 2007 ; accepted in final form 23 January 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
High-mobility group box 1 (HMGB1) released from necrotic cells or macrophages functions as a late inflammatory mediator and has been shown to induce cardiovascular collapse during sepsis. Thus far, however, the effect(s) of HMGB1 in the heart are not known. We determined the effects of HMGB1 on isolated feline cardiac myocytes by measuring sarcomere shortening in contracting cardiac myocytes, intracellular Ca2+ transients by using fluo-3, and L-type calcium currents by using whole cell perforate configuration of the patch-clamp technique. Treatment of isolated myocytes with HMGB1 (100 ng/ml) resulted in a 70% decrease in sarcomere shortening and a 50% decrease in the height of the peak Ca2+ transient within 5 min (P < 0.01). The immediate negative inotropic effects of HMGB1 on cell contractility and calcium homeostasis were partially reversible upon washout of HMGB1. A significant inhibition of the inward L-type calcium currents was also documented by the patch-clamp technique. HMGB1 induced the PKC-{varepsilon} translocation, and a PKC inhibitor significantly attenuated the negative inotropic effects of HMGB1. These studies show for the first time that HMGB1 impairs sarcomere shortening by decreasing calcium availability in cardiac myocytes through modulating membrane calcium influx and suggest that HMGB1 maybe acts as a novel myocardial depressant factor during cardiac injury.

inflammation; innate immunity; myocardial function; sepsis

MYOCARDIAL DYSFUNCTION, a central component in the complex pathophysiology of sepsis, contributes to the high mortality associated with this disorder. Indeed, in patients who develop cardiovascular impairment, the overall mortality rises from 20% to 70–90% (18). Lipopolysaccharide (LPS) depresses intrinsic myocardial contractility and is believed to be an important factor contributing to myocardial dysfunction during sepsis (17, 21). Although the endogenous agents that contribute to the myocardial depression observed in sepsis remain elusive, studies suggest that TNF, IL-1β, and nitric oxide (NO) play a role (1, 7, 13, 14).

High-mobility group box 1 (HMGB1) protein was originally described as a nuclear DNA-binding protein involved in the transcriptional regulation and gene expression. However, in 1999 Wang et al. (27) reported that activated macrophages secreted HMGB1. HMGB1 was subsequently shown to be released systemically in murine models of endotoxemia and sepsis induced by cecal perforation (28). Further supporting the importance of HMGB1 in the pathogenesis of sepsis was the observation that passive immunization with neutralizing anti-HMGB1 antibodies prevented organ dysfunction in septic mice (28). Recently, Gibot et al. (2) have shown that, as in experimental models of sepsis, HMGB1 plasma concentration correlates with the degree of organ dysfunction during septic shock. However, HMGB1 levels alone did not predict the outcome in this patient cohort.

Although previous studies have primarily addressed the role of myocardial TNF, IL-1β, and NO in the pathogenesis of sepsis-induced left ventricular dysfunction (1, 7, 13, 14), HMGB1 also is expressed in the heart following stress and therefore may contribute to the development of cardiac depression (22). In considering the cytokine properties of HMGB1 and its proposed role in sepsis-induced organ dysfunction, we sought to determine whether HMGB1 could directly depress myocyte contractility in vitro.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Effects of HMGB1 on cardiac myocyte function. All studies conformed with the principles of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Baylor College of Medicine Animal Care and Use Committee. The methods for isolating and culturing adult feline cardiac myocytes have been described in detail elsewhere (10). Sarcomere shortening and calcium transients were assessed in isolated contracting cardiac myocytes by using the IonOptix MyoCam system (IonOptix, Milton, MA). Freshly isolated cells were loaded with 10 µM fluo-3 AM (Invitrogen, Carlsbad, CA) in HEPES solution containing (in mM) 137 NaCl, 5.4 KCl, 1.8 CaCl2, 15 dextrose, 1.3 MgSO4, 1.2 NaH2PO4, and 20 HEPES (pH 7.4 with NaOH) at 37°C in the dark for 15 min. Fluo-3-loaded cells were then attached to coverslips precoated with 10 µg/ml laminin and mounted in a perfusion chamber (model no. RC 47 FSLP; Warner Instruments, Hamden, CT). Cells were then placed on the stage of an inverted microscope (OLYMPUS IX 70) and superfused with the same HEPES solution to remove unloaded dye. The excitation light was set at 485-nm wavelength, and emission was collected at 530-nm wavelength. The stage of the microscope was illuminated with red light (640–750 nm) through bright-field illumination optics to allow simultaneous measurements of fluorescence changes and cell shortening. The cells were paced at 0.5 Hz following a 2-min period of stabilization (baseline); the cells were superfused with diluent or HMGB1 (1–100 ng/ml; Sigma-Aldrich, St. Louis, MO). Sarcomere motion and fluorescence changes were recorded continuously by using IonOptix acquisition software, and the data were stored for an off-line analysis by IonWizard software (IonOptix 1999, version 5). The HMGB1 (Sigma-Aldrich) used in these studies was a full-length recombinant human protein that was expressed in Escherichia coli. In preliminary control studies, we established that purified LPS (0.25–25 µg/ml; Invivogen, San Diego, CA) had no effect on sarcomere shortening over a 30-min period (data not shown). In additional control studies, we treated the cells with 100 ng/ml of HMGB1 obtained from R&D Systems (Minneapolis, MN) as well HMGBiotech (Milano, Italy). These commercial preparations employed for these studies were also tested for endotoxin levels using the amebocyte lysate assay (Lonza, Basel, Switzerland).

Effects of HMGB1 on L-type calcium currents. L-type channel activity was measured as described (19). Briefly, freshly isolated adult feline cardiac myocytes were studied in a chamber mounted on an inverted microscope (Nikon, Tokyo, Japan) and were superfused with a normal physiological salt solution containing (in mmol/l) 145 NaCl, 4 KCl, 5 CsCl (to block potassium currents), 1 MgCl2, 2 CaCl2, 5 HEPES, 11 glucose (pH 7.4) at 37°C. Low-resistance (1–4 M{Omega}) patch pipettes were filled with a solution containing (in mmol/l) 100 cesium glutamate, 40 KCl, 1 MgCl2, 4 Na2-ATP, 0.5 EGTA, and 5 HEPES (pH 7.2) and 150 mg/ml nystatin. Experiments were initiated after sufficient access to the cytosol was achieved (access resistance < 15 M{Omega}; 10–20 min). Whole cell L-type Ca2+ currents were measured with 400-ms depolarizing test pulses from a holding potential of –40 mV (to inactivate sodium current). Junction potential was not corrected and was <10 mV. The cell capacitance was measured by using small hyperpolarizing test steps. Membrane potentials were controlled with an Axopatch 2B (Axon Instruments, Concord, ON, Canada) voltage-clamp amplifier by using pClamp8 (Axon Instruments) software and acquired with a Digidata 1200 analog digital converter (Axon Instruments). The data were analyzed with Clampfit (Axon Instruments) and presented with Origin 6.0 (MicroCal Software, Northampton, MA). Only myocytes with minimal (<10%) rundown of L-type calcium current were included in the data analysis.

Effects of HMGB1 on PKC-{varepsilon} translocation. Freshly isolated feline ventricular myocytes were cultured in medium 199 supplemented with 0.1% human serum albumin and 1% insulin, transferrin, and selenium (ITS, Cellgro) for 6 h. The culture medium was then aspirated and the cells suspended in HEPES solution (described in Effects of HMGB1 on cardiac myocyte function) and were then exposed to HMGB1 (100 ng/ml) for 1, 3, and 10 min. The cells were lysed by adding 0.3 ml lysis buffer containing 50 mM NaF, 2 mM EGTA, 2 mM EDTA, 500 µM sodium orthovanadate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 0.5 mM phenylmethylsulfonyl fluoride, and 20 mM Tris (pH 7.4). Cell lysates were centrifuged at 100,000 g for 1 h at 4°C, and the supernatant was saved as the cytosolic fraction. The pellet was then resuspended in lysis buffer containing 1% Triton X-100 and sonicated. The resuspended pellets were incubated in a shaking ice bath for 15 min, centrifuged at 14,000 g for 10 min, and the supernatant saved as the membrane fraction. Aliquots of the cytosolic and membrane fractions were subjected to electrophoresis and immunoblotting for PKC-{varepsilon} using a mouse monoclonal antibody (1:2,000; BD Biosciences, San Jose, CA). Translocation of PKC-{varepsilon} was defined as the ratio of membrane fraction to cytosolic fraction.

Effects of PKC inhibition on the functional effects of HMGB1. To determine whether PKC-{varepsilon} inhibition was sufficient to attenuate the effects of HMGB1 (100 ng/ml), freshly isolated cardiac myocytes were pretreated for 10 min with Ro-31-8220 (0.001–1 µM; Calbiochem, San Diego, CA), a PKC inhibitor, prior to assessing sarcomere shortening as described in Effects of HMGB1 on cardiac myocyte function.

Effects of receptor for advanced glycation end products and TLR4 antibodies on the functional effects of HMGB1. To determine whether the negative inotropic effects of HMGB1 were mediated by activation of Toll-like receptor 4 (TLR4) and/or the receptor for advanced glycation end products (RAGE), freshly isolated cardiac myocytes were pretreated for 30 min with an anti-RAGE antibody (10 µg/ml; R&D Systems) or anti-TLR4 antibody (10 µg/ml; clone HTA125; Gene Tex, San Antonio, TX) before stimulation with HMGB1 (100 ng/ml) as described in Effects of HMGB1 on cardiac myocyte function.

Statistical analysis. All data are expressed as the means ± SE. Statistical significance was evaluated by two-way ANOVA. A post hoc test of least significant differences (Bonferroni or Dunnett) was used to determine differences among groups where appropriate. A probability value of P < 0.05 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of HMGB1 on cardiac myocyte function. Following an initial period of stabilization (2 min), freshly isolated adult feline cardiac myocytes were superfused with diluent or 100 ng/ml of HMGB1. Figure 1, A and B, depicts continuous tracings of sarcomere shortening (top) and calcium transients (bottom) for diluent treated cells, whereas Fig. 1, C and D, depicts tracings of sarcomere shortening and calcium transients in HMGB1-treated cells. As shown, treatment with diluent had no significant effect on sarcomere motion or the peak calcium transients during the period of observation. In contrast, treatment with HMGB1 resulted in a rapid (within 5 min) decrease in sarcomere shortening that was accompanied by a decrease in the peak amplitude of the calcium transient. Importantly, the effects of HMGB1 (Fig. 1B) were partially reversible following washout of HMGB1 from the superfusate. Figure 2 summarizes the results of group data. Treatment with 100 ng/ml of HMGB1 resulted in a significant (P < 0.01) 70% decrease in sarcomere shortening, which was accompanied by a significant (P < 0.01) 50% decrease in peak fluorescence brightness. The effects of HMGB1 on sarcomere shortening and peak fluorescence brightness were partially reversible during the washout phase of the experiment. Sarcomere shortening following washout of HMGB1 did not differ significantly from baseline. To determine whether the effects of HMGB1 (Sigma) on sarcomere shortening were spurious, that is, secondary to either an artifact of the commercial preparation and/or endotoxin contamination of the recombinant protein, we repeated the above experiments using two additional commercially available preparations. As shown in Table 1, all of the preparations of HMGB1 studied provoked a significant decrease in sarcomere shortening in isolated contracting cardiac myocytes. Furthermore, concentrations of endotoxin that overlapped those found in the HMGB1 preparations did not affect sarcomere shortening following a 10–30-min exposure (data not shown), consistent with the repeated observation that effects of endotoxin on isolated cardiac myocytes are time dependent and require ~6 h to become manifest.


Figure 1
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  		Fig. 1. Effects of high-mobility group box 1 (HMGB1) on cardiac myocyte contractility and intracellular calcium transients. Recordings of myocyte contractility during the entire period of experimentation in individual cells treated with diluent (A) or HMGB1 (100 ng/ml, B). Vertical axis shows absolute sarcomere length in micrometers or fluorescence brightness (in arbitrary units). The horizontal axis represents time (in seconds). Representative pacing events recorded under baseline (C) and posttreatment with HMGB1 (100 ng/ml, D). Vertical axis represents fluorescence brightness in arbitrary units.

 

Figure 2
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  		Fig. 2. Effects of HMGB1 on cardiac myocyte contractility and peak amplitude of calcium transients. A: effect of 100 ng/ml HMGB1 on the sarcomere shortening. B: effect of 100 ng/ml HMGB1 on the peak fluorescence brightness (*P < 0.01 vs. baseline; n = 9 cells from 4 hearts).

 

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  		Table 1. Effect of different preparations of HMGB1 proteins on myocyte contractility

 
To determine whether the effects of HMGB1 were dose dependent, we treated isolated contracting cardiac myocytes with HMGB1 concentrations ranging from 1–100 ng/ml. Figure 3, A and B, summarizes the effects of different HMGB1 concentrations on sarcomere shortening and the peak amplitude of calcium transients, respectively. As shown, at concentrations ≥10 ng/ml, HMGB1 produced a significant (P < 0.01 compared with diluent) decrease in sarcomere shortening and peak amplitude of calcium transients. Thus pathophysiological concentrations of HMGB1 are sufficient to produce negative inotropic effects and depress calcium transients in isolated contracting cardiac myocytes.


Figure 3
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  		Fig. 3. Dose-dependent effects of HMGB1 on myocyte contractility and intracellular calcium transients. A: effects of increasing concentrations of HMGB1 on sarcomere shortening. B: effects of increasing concentrations of HMGB1 on the peak amplitude of calcium transients (*P < 0.01 vs. diluent; n = 8 cells/condition). Exp, experimental (HMGB1).

 
Effects of HMGB1 on L-type calcium currents. To determine the mechanisms for the negative inotropic effects of HMGB1, we evaluated L-type calcium channel activity in diluent and HMGB1-treated adult cardiac myocytes. Figure 4, A and B, depicts the effect on the HMGB1 on the inward calcium current. As shown by the representative tracings in Fig. 4A, exposure to HMGB1 (10 ng/ml) dramatically reduced the inward L-type calcium current. A partial recovery of channel activity was noted after washout of HMGB1. Figure 4B, which summarizes the results of group data, shows that HMGB1 (10 ng/ml) led to a significant (P < 0.01) inhibition (70%) of the inward calcium current in isolated myocytes and that this was partially reversible following washout. Time-course experiments (Fig. 4C) revealed that the effect of HMGB1 occurred within 1 to 2 min and reached plateau within 5 min after challenge. These latter findings are in agreement with the effects of HMGB1 on myocyte contractility and peak amplitude of intracellular calcium transients shown in Fig. 2, A and B.


Figure 4
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  		Fig. 4. Effect of HMGB1 on L-type calcium currents. A: inward L-type calcium currents recorded under baseline conditions, after HMGB1 treatment, and washout. B: relative effects of HMGB1 (10 ng/ml) on the L-type calcium currents in isolated myocytes. C: time course of the L-type calcium currents in the presence and after washout of HMGB1 (n = 8 cells/condition). ICa, inward calcium current.

 
Effect of HMGB1 on PKC-{varepsilon} translocation. Activation of PKC-{varepsilon} has been linked to the inhibition of L-type calcium channel activity in mammalian cardiac myocytes (3). Accordingly, we examined the effects of HMGB1 (100 ng/ml) on PKC-{varepsilon} activation in isolated feline cardiac myocytes. Figure 5A depicts representative Western blot analysis for cytosolic and membrane PKC-{varepsilon} fractions in the presence and absence of HMGB1, whereas Fig. 5B summarizes the results of group data. As shown, HMGB1 induced a significant (P < 0.01) translocation of PKC-{varepsilon} from the cytosol to the membrane within 10 min, consistent with the rapid activation of the L-calcium currents in the whole cell patch-clamp studies (Fig. 4C).


Figure 5
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  		Fig. 5. HMGB1 induces PKC-{varepsilon} translocation in cardiac myocytes. A: representative Western blot analysis for cytosolic and membrane PKC-{varepsilon}. HMGB1 (100 ng/ml) induced PKC-{varepsilon} translocation from the cytosol (Cyto) to the membrane fraction (Mem). B: time course of PKC-{varepsilon} translocation in cardiac myocytes after exposure to HMGB1 (100 ng/ml) (*P < 0.01 vs. time 0, n = 4 isolations). C: Ro-31-8220 significantly attenuated the effects of HMGB1 (100 ng/ml) in a dose-dependent manner compared with diluent; n = 10–15 cells/condition (*P < 0.01 vs. diluent; {ddagger}P < 0.05 vs. HMGB1 alone).

 
To determine whether inhibition of PKC translocation was sufficient to prevent the effects of HMGB1 in cardiac myocytes, we pretreated the cells with varying concentrations of the PKC inhibitor Ro-31-8220 (0.001–1 µM). Figure 5C summarizes the effects of Ro-31-8220 on sarcomere shortening. Ro-31-8220 had no effect on sarcomere shortening at baseline but did significantly attenuate the deleterious effects of HMGB1 on sarcomere shortening in a dose-dependent manner (P < 0.01 compared with HMGB1 diluent-treated cells).

RAGE and TLR4 mediate the negative inotropic effects of HMGB. Previously, we have shown that the negative inotropic effects of HMGB1 were not mediated via Toll-like receptor 2 (TLR2) (22). HMGB1 has been reported to bind to and signal through the multivalent immunoglobulin receptors RAGE and TLR4 (12) (16). To determine whether the RAGE and/or TLR4 receptors mediated the negative inotropic effects of HMGB1, we performed studies using inhibitory antibodies against RAGE and TLR4. As shown in Fig. 6A, a neutralizing anti-RAGE antibody significantly attenuated the negative inotropic effects of HMGB1 in isolated cardiac myocytes. Specifically, pretreatment with a RAGE antibody resulted in a 32% increase in the extent of sarcomere shortening in HMGB1-treated cells, compared with cells that had been pretreated with diluent alone. Figure 6B shows that the TLR4 antibody also had a significant effect on sarcomere shortening following exposure to HMGB1. Pretreatment with TLR4 antibody resulted in a significant 22% improvement in sarcomere shortening compared with HMGB1-stimulated cells that had been pretreated with diluent alone. Taken together, these data suggest that the negative inotropic effect of HMGB1 on cardiac myocytes is, at least in part, mediated by binding to both RAGE and TLR4.


Figure 6
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  		Fig. 6. Effects of anti-receptor for advanced glycation end products (RAGE) and Toll-like receptor 4 (TLR4) antibodies on HMGB1-induced negative inotropic effects in cardiac myocytes. Cells were preincubated (30 min) with each antibody before exposure to HMGB1 (100 ng/ml). A: RAGE blockade improved sarcomere shortening by 32%. B: TLR4 blockade improved sarcomere shortening by 22% (*P < 0.01 vs. diluent; {ddagger}P < 0.05 vs. HMGB1 alone; n = 10–15 cells/condition).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The results of this study show for the first time that pathophysiologically relevant concentrations of HMGB1 are sufficient to produce negative inotropic effects in isolated contracting cardiac myocytes through a mechanism that involves inhibition of the L-type calcium channel. Several lines of evidence support this statement. First, treatment with HMGB1 produced a rapid decrease in sarcomere shortening that was accompanied by a decrease in the peak amplitude of the intracellular calcium transient in isolated contracting cardiac myocytes (Figs. 1 and 2). The effects of HMGB1 on sarcomere shortening and calcium homeostasis were dose dependent (Fig. 3) and were partially reversible following washout of HMGB1 (Fig. 2). It is unlikely that the observed negative inotropic effects of HMGB1 were secondary to LPS contamination, insofar as the HMGB1 used in this study contained 25 ng LPS per 1,000 ng HMGB1, which is not sufficient to provoke negative inotropic effects within the time course that we observed negative inotropic effects with HMGB1. Indeed, in preliminary control studies we determined that purified LPS (0.25–25 µg/ml) had neither an effect on cell shortening nor an effect on the peak amplitude of intracellular calcium transients over a 30-min period (data not shown), consistent with prior observations (6, 8, 11, 15, 20, 24, 25). Furthermore, when we used two additional commercially available preparations of HMGB1, each of which had lower concentrations of contaminating endotoxin (0.4–10 µg/ml), we still observed significant decreases in sarcomere shortening. Of note, the magnitude of the effect of HMGB1 on sarcomere shortening exceeded the magnitude of the decrease in amplitude of the peak calcium transient, suggesting that HMGB1 may impair sarcomere shortening through calcium-independent mechanisms. Alternatively, HMGB1 may provoke decreased calcium sensitivity in cardiac myocytes. Additional studies will be required to address this important question. Second, treatment with HMGB1 led to a significant decrease in the inward L-type calcium current (Fig. 4). Importantly, time-course experiments (Fig. 4C) revealed that the effect of HMGB1 was manifest within 1 to 2 min and reached plateau by 5 min after HMGB1 provocation, consistent with the rapid time course of the effects of HMGB1 on the peak inward calcium transient. Taken together, these studies show that HMGB1 exerts dose-dependent negative inotropic effects in cardiac myocytes by decreasing the amount of activator calcium from the inward calcium that is necessary for calcium-triggered calcium release and suggest that HMGB1 maybe acts as a novel myocardial depressant factor during cardiac injury. Although the scope of this study was not intended to delineate the full spectrum of mechanisms for the deleterious negative inotropic effects of HMGB1, our results do suggest that HMGB1-induced activation of PKC-{varepsilon} is responsible, at least in part, for the negative inotropic effects of HMGB1 in cardiac myocytes; that is, HMGB1 induced the rapid translocation of PKC-{varepsilon} from the cytosol with a time course that was consistent with the inhibition of the L-type calcium channel, as well as the decrease in sarcomere shortening and peak intracellular calcium transients. Indeed, Hu et al. (3) have shown that activation of PKC-{varepsilon} by a specific agonist ({varepsilon}-V1–7) inhibits the L-type calcium channel activity in adult rat ventricular myocytes. Moreover, the negative inotropic effects of HMGB1 were sensitive to Ro-31-8220, a PKC inhibitor (Fig. 5C).

HMGB1 and tissue injury. Although HMGB1 was originally described as a late mediator of inflammation and endotoxin lethality (27), more recent studies have shown that HMGB1 plays an important role as an early inflammatory mediator following acute tissue injury. For example, Kim et al. (4) demonstrated increased HMGB1 expression in the lung within 4 h of inducing hemorrhagic shock. These authors showed that an administration of anti-HMGB1 antibody reduced the accumulation of neutrophils in the lung as well as lung permeability. Similarly, HMGB1 levels were increased within 1 h in ischemia-reperfusion (I/R) injury of the liver. Pertinent to the present discussion, administration of recombinant HMGB1 to mice that were undergoing partial hepatic I/R led to worsening outcomes. Moreover, a neutralizing HMGB1 antibody decreased hepatic damage following I/R injury (26). Although the present results are consistent with prior observations that the acute effects of HMGB1 may be deleterious, it is interesting to note that the exogenous administration of HMGB1 has been reported to be beneficial in a mouse model of myocardial infarction by inducing myocardial regeneration and improving cardiac function (9). Interestingly, HMGB1 has also been linked to increased angiogenesis (23). Taken together, these latter findings raise the interesting possibility that, in a more chronic model of inflammation, HMGB1 may play an important role in promoting cardiac repair and/or cardiac regeneration.

Previous studies have shown that mice with defective TLR4 signaling are protected against HMGB1-induced hepatic I/R injury, suggesting that TLR4 serves as a receptor for HMGB1 (26). However, other studies have shown that HMGB1 signaling can also be mediated via TLR2 and/or RAGE (5, 16). Previously, we have shown the HMGB1 provoked negative inotropic effects in cardiac myocytes isolated from wild-type and TLR2-deficient mice, suggesting that TLR2 is not necessary for mediating the negative inotropic effects of HMGB1 (22). Here we show for the first time that the deleterious effects of HMGB1 on sarcomere shortening are attributable, at least in part, to the activation of RAGE and TLR4 (Fig. 6); that is, the effects of HMGB1 were partially inhibited by pretreatment with neutralizing antibodies against RAGE and TLR4. We and others (6, 8, 11, 15, 20, 24, 25) have shown that LPS, the canonical TLR4 ligand, produces negative inotropic effects after 1–6 h of stimulation, suggesting that LPS-induced negative inotropic effects require de novo synthesis of one or more different biological mediators. In contrast, in the present study the effects of HMGB1 on sarcomere motion and calcium homeostasis were apparent within minutes. At present, it is not apparent why HMGB1 and LPS, both of which signal through TLR4, have different time courses for producing negative inotropic effects in cardiac myocytes. One possibility, albeit speculative, is that there may be cross talk between the RAGE and TLR4 receptors, which might explain the more rapid onset on negative inotropic effects with HMGB1. Further studies will be necessary to address this interesting question.

In conclusion, the findings of the present study suggest that HMGB1 may act as a novel myocardial depressant factor that is released by resident myocardial cells following tissue injury. Although substances that produce negative inotropic effects have traditionally been viewed as deleterious, it is possible that locally released HMGB1 may decrease energy utilization in ischemic tissue, thereby preventing injured myocytes from worsening ATP depletion that eventuate in necrosis. This point of view is consistent with the aforementioned possibility that sustained HMGB1 signaling may play an important role in cardiac repair and/or regeneration (9, 23). This statement notwithstanding, it is likely that excessive release of HMGB1 acutely may be overtly deleterious to the heart by contributing to excessive and/or sustained inflammation and/or profound myocardial depression and myocardial collapse.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported by National Institutes of Health Grants HL-58081, HL-42250, HL-073017 (to D. L. Mann), and GM-62474 (to J. G. Vallejo).


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. L. Mann, Winters Ctr. for Heart Failure Research, 1709 Dryden-BCM, 620-Rm. 9.83, Houston, TX 77030 (e-mail: dmann{at}bcm.tmc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* H.-P. Tzeng and J. Fan contributed equally to this work. Back


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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