This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sibelius, U. Articles by Grimminger, F. Search for Related Content PubMed PubMed Citation Articles by Sibelius, U. Articles by Grimminger, F. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB ACETYLSALICYLIC ACID INDOMETHACIN POTASSIUM Related Collections Contractile function Other myocardial biology Other heart failure Animal models of human disease

(Circulation. 2000;101:78.)
© 2000 American Heart Association, Inc.

Basic Science Reports

Staphylococcal -Toxin Provokes Coronary Vasoconstriction and Loss in Myocardial Contractility in Perfused Rat Hearts

Role of Thromboxane Generation

Ulf Sibelius, MD; Ulrich Grandel, MD; Michael Buerke, MD; Doris Mueller; Ladislau Kiss, PhD; Hans-Joachim Kraemer, MD; Ruediger Braun-Dullaeus, MD; Werner Haberbosch, MD; Werner Seeger, MD; Friedrich Grimminger, MD, PhD

From the Department of Internal Medicine, Justus-Liebig-University, Giessen, and the II Department of Internal Medicine, Johannes-Gutenberg-University, Mainz (M.B.), Germany.

Correspondence to F. Grimminger, MD, PhD, Department of Internal Medicine, Klinikstrasse 36, D-35392 Giessen, Germany. E-mail friedrich.grimminger{at}innere.med.uni-giessen.de

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background—Cardiac performance is severely depressed in septic shock. Endotoxin has been implicated as the causative agent in Gram-negative sepsis, but similar abnormalities are encountered in Gram-positive sepsis. We investigated the influence of the major exotoxin of Staphylococcus aureus, staphylococcal -toxin, in isolated perfused rat hearts.

Methods and Results—-Toxin 0.25 to 1 µg/mL caused a dose-dependent increase in coronary perfusion pressure that more than doubled. In parallel, we noted a decrease in left ventricular developed pressure and the maximum rate of left ventricular pressure rise (dP/dt_max), dropping to a minimum of <60% of control. These changes were accompanied by a liberation of thromboxane A₂ and prostacyclin into the coronary effluent. The release of creatine kinase, lactate dehydrogenase, potassium, and lactate did not surpass control heart values, and leukotrienes were also not detected. Indomethacin, acetylsalicylic acid, and the thromboxane receptor antagonist daltroban fully blocked the -toxin–induced coronary vasoconstrictor response and the decrease in left ventricular developed pressure and dP/dt_max, whereas the lipoxygenase inhibitor nordihydroguaiaretic acid, the platelet activating factor antagonist WEB 2086, and the -adrenergic antagonist phentolamine were entirely ineffective. Inhibition of nitric oxide synthase even enhanced the -toxin–induced increase in coronary perfusion pressure and the loss in myocardial performance.

Conclusions—Purified staphylococcal -toxin provokes coronary vasoconstriction and loss in myocardial contractility. The responses appear to be largely attributable to the generation of thromboxane and are even enhanced when the endogenous nitric oxide synthesis is blocked. Bacterial exotoxins, such as staphylococcal -toxin, may thus be implicated in the loss of cardiac performance encountered in Gram-positive septic shock.

Key Words: vasoconstriction • contractility • toxins

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
In addition to vascular hyporeactivity, progressive myocardial depression is a characteristic feature of cardiocirculatory changes of septic shock and contributes to the high mortality of this disease. Despite an elevated cardiac output, myocardial performance is deteriorated, as indicated by reduced left and right ventricular ejection fractions and dilatation of both ventricles.^{1 2 3 4}

Circulating myocardial depressant agents have been implicated in these findings; endotoxin and secondarily induced cytokines, such as tumor necrosis factor (TNF)- and interleukin (IL)-1, are major culprits in this context.^{5 6 7 8 9} Both endotoxin per se and TNF- are potent inducers of the inducible nitric oxide (NO) synthase in the myocardium, and excessive endogenous NO synthesis may depress myocardial contractile performance. Conversely, NO is known to play a basic role in the regulation of myocardial blood flow, and inhibition of NO synthesis caused myocardial ischemia in endotoxemic rats.¹⁰ This does not necessarily require a reduction of global myocardial perfusion, but microcirculatory disturbances leading to insufficient regional oxygen availability may be responsible for this phenomenon.^{10 11}

There is growing evidence that the coronary circulation in sepsis is susceptible to a maldistribution of regional blood flow that may contribute to myocardial failure. Histological changes in the myocardium of septic sheep were compatible with focal ischemia on the microcirculatory rather than systemic level.¹² In endotoxic dogs, disturbances of coronary microcirculation were associated with depressed contractility,¹³ and septic sheep were recently noted to be unable to sufficiently augment myocardial blood flow in response to increased O₂ demands.¹⁴

The typical pattern of cardiovascular dysfunction, however, is also encountered in Gram-positive septic shock.¹⁵ In a canine model of septic shock, intraperitoneal application of Escherichia coli or Staphylococcus aureus provoked myocardial depression, but, as anticipated, endotoxemia was discovered only with E coli.¹⁶ The predominant toxin of S aureus is the -toxin, the prototype of pore-forming exotoxins from Gram-positive rods, and purified -toxin provoked cardiovascular collapse in intact animals¹⁷ and suppressed tension development in isolated rat atria in vitro.¹⁸ Although the problem of quantification of exotoxins in biological samples is not fully resolved, because of the very rapid membrane incorporation of this agent, experimental data suggest that small numbers of exotoxins suffice to activate target cells.^{19 20} Following this line, we now investigated the impact of -toxin on cardiac function in the absence of endotoxin, serum, and circulating inflammatory cells in isolated perfused rat hearts. We found that the -toxin is a potent coronary vasoconstrictor, and the pressure response is accompanied by a marked depression of myocardial contractility. Interestingly, both mediator analysis and pharmacological intervention strongly indicated that the abnormalities in cardiac performance can be attributed largely to the toxin-elicited thromboxane (Tx) formation. In view of the fact that -toxin is a representative of a large family of pore-forming exotoxins originating from Gram-positive but also Gram-negative bacteria, the present findings suggest that these bacterial agents should be considered to be contributors to cardiac abnormalities in septic shock.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Materials
Purified -toxin from S aureus proven to be endotoxin-free was provided by S. Bhakdi, MD (Department of Medical Microbiology, Mainz, Germany). Salmonella abortus equii lipopolysaccharide (LPS) was obtained from C. Gallanos, PhD (Max Planck Institute for Immunology, Freiburg, Germany). Indomethacin was purchased from ICN Biomedicals Inc and the TxA₂ receptor antagonist daltroban (BM 13.505) from Boehringer. The -adrenergic antagonist phentolamine, the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA), and acetylsalicylic acid were purchased from Sigma; the NO synthase inhibitor N^G-monomethyl-L-arginine (L-NMMA) from Calbiochem; and the platelet-activating factor antagonist WEB 2086 from Boehringer.

Preparation and Isolated Heart Perfusion
Male Wistar rats (Charles River, Sulzfeld, Germany) were heparinized (heparin 1000 IU/kg) and anesthetized (pentobarbitone 60 mg/kg) by intraperitoneal injection. The hearts were rapidly excised and immersed in ice-cold Krebs-Henseleit buffer solution (KHBS). After the organ weight had been determined, the hearts were attached to a Langendorff perfusion apparatus. The hearts were retrogradely perfused at a constant flow (10 mL · min^-1 · g^-1) with a modified KHBS containing (in mmol/L) NaCl 125, KCl 4.3, KH₂PO₄ 1.1, MgCl₂ · 6H₂O 1.3, CaCl₂ · 2H₂O 2.4, NaHCO₃ 25, and glucose 13.32. The perfusate was gassed with carbogen (5% CO₂/95% O₂). The pH was 7.4±0.03, PO₂ 500±45 mm Hg, and PCO₂ 35±5 mm Hg at 37°C. All hearts were initially rinsed with 150 mL KHBS in a nonrecirculating mode before switching to recirculation (total volume 50 mL).

For monitoring coronary perfusion pressure (CPP), the aortic cannula was connected to a pressure transducer (Braun). To measure left ventricular contractility, a latex balloon attached to a second pressure transducer was inserted into the left ventricular cavity. Left ventricular developed pressure (LVDP) was calculated as the difference between peak-systolic and end-diastolic pressure (8 to 12 mm Hg), and the maximum rate of left ventricular pressure rise (dP/dt_max) was computed by a differentiator (Schwarzer DRE 48, Picker). The hearts were paced at 320 to 360 bpm by a Stimulator P Type 201 (Hugo Sachs Elektronik). All physiological parameters were continuously recorded on a 12-channel polygraph (Schwarzer CU 12-N, Picker). At the end of each experiment, the heart weight was measured again, and the magnitude of edema formation was calculated as the difference between heart weight before and after perfusion.

Experimental Protocols
After the hearts had been equilibrated for 20 minutes, time was set to zero, and staphylococcal -toxin was admixed to the perfusate at final concentrations of 0.25 (n=6), 0.5 (n=9), and 1 (n=6) µg/mL. Physiological variables were monitored for 120 minutes. Perfusate samples were taken twice before as well as 10, 20, 40, 60, 90, and 120 minutes after toxin application. For pharmacological intervention, either indomethacin 100 µmol/L (n=6), acetylsalicylic acid 500 µmol/L (n=4), daltroban 10 µmol/L (n=4), NDGA 5 µmol/L (n=3), WEB 2086 10 µmol/L (n=3), L-NMMA 25 µmol/L (n=4), or phentolamine 5 µmol/L (n=4) was preadmixed to the perfusate after the recirculating perfusion was begun. In these experiments, -toxin was used at a concentration of 0.5 µg/mL. Control experiments included the perfusion with only buffer fluid (n=8) and with buffer fluid enriched with the respective pharmacological inhibitors (n=3). In a separate type of study, LPS 100 ng/mL was admixed to the recirculating medium for 1 hour, followed by administration of -toxin 0.5 µg/mL (n=3) or sham application of exotoxin (n=3). Further studies addressing -toxin–induced morphological changes used a 1-hour and a 2-hour perfusion period in the presence of 1 µg/mL exotoxin and in the absence of inhibitors (n=3). Additional experiments using 0.5 µg/mL -toxin were terminated after 40 minutes (n=4) to determine the increase in heart weight at this time point.

Histological Analysis of Tissue Injury and of Residual Cells in the Myocardium
After the perfusion was stopped, the coronary vasculature was perfused with a fixative (paraformaldehyde 4% in PBS, pH 7.4). Thin slices of the left ventricular free wall were placed in the fixative at 4°C for 1.5 hours, followed by dehydration in a graded series of acetone solutions (4°C). Tissue blocks were embedded in Immunobed (Polyscience Inc) at 4°C for 12 hours. Sections 5 µm thick were cut, transferred to coated slides, and stained with hematoxylin/eosin solution. The sections were examined at a magnification of x400 or x250 to determine the presence of adhering and infiltrating neutrophils, eosinophils, basophils, monocytes, lymphocytes, and platelets. The total number of cells was analyzed in 10 separate fields for each tissue section and expressed as cells/mm².

Biochemical Assays
TxA₂, prostacyclin (PGI₂), and TNF- were measured by commercially available ELISAs (Cayman Chemical Co; Biosource). Leukotrienes (LTs) (LTB₄, LTC₄, LTD₄, and LTE₄) were analyzed by use of previously described chromatographic techniques.²¹ Lactate dehydrogenase (LDH), creatine kinase (CK), lactate, and potassium (K⁺) were measured by routine techniques.

Statistical Analysis
All data are given as mean±SEM. Data were analyzed by 1-way ANOVA followed by Tukey’s honestly significant difference test when differences among groups were to be determined. A value of P<0.05 was considered to be significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Whereas control hearts displayed stable values of CPP over the observation period, -toxin caused a rapid, dose-dependent increase in CPP (Figure 1). At the highest dose (1 µg/mL), CPP more than doubled within <20 minutes, and almost equally high plateau values were achieved with 0.5 µg/mL -toxin. With corresponding dose- and time-dependence, myocardial contractility was depressed in response to -toxin (Figures 2 and 3). Within 20 minutes, a decline of LVDP and dP/dt_max to values <60% below baseline occurred in the presence of 1 µg/mL -toxin, and 0.5 µg/mL resulted in a loss of contractility of 15%. Even the myocardial depression caused by the lowest dose of toxin (0.25 µg/mL) differed from control. In addition, -toxin provoked an increase in heart weight, which nearly reached its maximum after 40 minutes (Figure 4). These changes in heart physiology were accompanied by a release of TxB₂ and 6-keto-PGF₁, the stable metabolites of TxA₂ and prostacyclin, into the perfusate (Figure 5). Markers of myocardial cell necrosis (CK, LDH, K⁺) and lactate accumulated in the perfusate to some minor extent, but no significant difference between control and -toxin–challenged hearts was noted (Table). Cysteinyl-LTs (LTC₄/LTD₄/LTE₄) and LTB₄ were not detected in response to administration of -toxin (data not shown).

View larger version (25K): [in this window] [in a new window]   Figure 1. CPP after application of -toxin 0.25 µg/mL (), 0.5 µg/mL (), and 1 µg/mL () and control experiments (). Mean±SEM of 6 experiments each is given. Values marked * and the following differ significantly from control.

View larger version (27K): [in this window] [in a new window]   Figure 2. LVDP after application of -toxin 0.25 µg/mL (), 0.5 µg/mL (), and 1 µg/mL () and control experiments (). Mean±SEM of at least 6 experiments each is given. Values marked * and the following differ significantly from control.

View larger version (27K): [in this window] [in a new window]   Figure 3. Influence of -toxin on dP/dtmax in response to -toxin 0.25 µg/mL (), 0.5 µg/mL (), 1 µg/mL (), and control (). Mean±SEM of 6 experiments each is given. Values marked * and the following differ significantly from control.

View larger version (33K): [in this window] [in a new window]   Figure 4. Edema formation measured after 40 and 120 minutes as gain in heart wet weight. -Toxin was used at 0.5 µg/mL. Mean±SEM of 4 experiments each is given.

View larger version (20K): [in this window] [in a new window]   Figure 5. Liberation of vasoactive prostanoids TxA2 (designated as TxB2, stable hydrolysis product of TxA2), top, and prostacyclin (designated as 6-keto-PGF1, stable hydrolysis product of prostacyclin), bottom, into perfusate after application of -toxin 0.5 µg/mL () and in presence of indomethacin 100 µmol/L (), daltroban 10 µmol/L (, top panel only) or phentolamine 5 µmol/L (x, top panel only). Control experiments () were performed in absence of -toxin. Mean±SEM of at least 4 experiments is given. Values marked with * and the following differ significantly from control.

View this table: [in this window] [in a new window]   Table 1. Release of CK, LDH, K+, and Lactate in Response to -Toxin in the Absence and Presence of Inhibitors

In the presence of indomethacin, the -toxin–induced increase in CPP was totally blocked (Figure 6), and the loss of myocardial performance was completely abolished (Figures 7 and 8). This was also true using acetylsalicylic acid (data not shown). Neither TxB₂ nor 6-keto-PGF₁ was detected under these conditions (Figure 5). Indomethacin did, however, not affect the -toxin–induced increase in heart weight (Figure 4). The release of CK, LDH, K⁺, and lactate was not changed in the presence of the cyclooxygenase inhibitor (Table).

View larger version (23K): [in this window] [in a new window]   Figure 6. Influence of indomethacin 100 µmol/L, daltroban 10 µmol/L, phentolamine 5 µmol/L, and L-NMMA 25 µmol/L on -toxin 0.5 µg/mL–induced increase of CPP after 45 minutes. Control experiments using identical inhibitor concentrations were performed in absence of -toxin. In these control studies (not shown in detail), no influence of indomethacin, daltroban, phentolamine, or L-NMMA on baseline CPP was noted. Mean±SEM of at least 4 experiments each is given. *Significant difference from control. **Significant difference from control and -toxin.

View larger version (26K): [in this window] [in a new window]   Figure 7. Influence of indomethacin 100 µmol/L, daltroban 10 µmol/L, phentolamine 5 µmol/L, and L-NMMA 25 µmol/L on -toxin 0.5 µg/mL–induced depression of LVDP after 45 minutes. Control experiments using identical inhibitor concentrations were performed in absence of -toxin. In these control studies (not shown in detail), no influence of indomethacin, daltroban, phentolamine, or L-NMMA on baseline LVDP was noted. Mean±SEM of 4 experiments each is given. *Significant difference from control.

View larger version (29K): [in this window] [in a new window]   Figure 8. Influence of indomethacin 100 µmol/L, daltroban 10 µmol/L, phentolamine 5 µmol/L, and L-NMMA 25 µmol/L on -toxin 0.5 µg/mL–induced depression of dP/dtmax after 45 minutes. Control experiments using identical inhibitor concentrations were performed in absence of -toxin. In these control studies (not shown in detail), no influence of indomethacin, daltroban, phentolamine, or L-NMMA on baseline dP/dtmax was noted. Mean±SEM from 4 independent experiments each is given. *Significant difference from control.

Similarly, the specific TxA₂ receptor antagonist daltroban fully blocked the rise in CPP and the depression of contractility (Figures 6, 7, and 8), whereas the synthesis of TxB₂ was not affected (Figure 5). However, there was some decrease in -toxin–induced heart weight gain in the presence of daltroban (Figure 4). In contrast, neither the lipoxygenase inhibitor NDGA, the platelet-activating factor receptor antagonist WEB 2086 (data not shown), nor the -adrenergic receptor antagonist phentolamine affected the -toxin–elicited coronary vasoconstriction and the loss in myocardial performance (Figures 6, 7, and 8). In the presence of the NO synthase inhibitor L-NMMA, the -toxin–elicited increase in CPP and the decrease in contractile performance were even markedly enhanced (Figures 6, 7, and 8).

Staphylococcal -toxin did not provoke release of TNF- into the perfusate. Administration of LPS did not provoke any significant changes in CPP, LVDP, or dP/dt_max. Moreover, previous LPS administration did not influence the vasoconstriction and the loss in myocardial performance induced by a subsequent -toxin challenge (data not shown).

Histological examination of control hearts did not show any morphological abnormality. This was also true for hearts exposed to -toxin 1.0 µg/mL for 1 and 2 hours, except for some myofibrillar contraction bands indicating a minor morphologically detectable injury (Figure 9). Because leukocytes and platelets might adhere to the coronary endothelium before excision of the hearts and thus might contribute to the prostanoid synthesis, we analyzed the number of these cell types in isolated hearts before onset of toxin challenge. The number of each leukocyte type or thrombocytes ranged below 2 cells/mm², thus excluding any significant contribution to the overall mediator generation.

View larger version (77K): [in this window] [in a new window]   Figure 9. Photomicrographs of myocardial sections of Langendorff rat hearts treated with either vehicle (left) or 1 µg/mL -toxin for 2 hours. Left, Myocardial tissue from a Langendorff rat heart subjected to 2 hours of perfusion and treated with vehicle. Normal cardiac myocytes with cross-striations. Right, Myocardial tissue from a Langendorff rat heart subjected to 2 hours of perfusion and treated with 1 µg/mL -toxin. Cardiac myocytes demonstrated slight injury, with loss of cross-striation and contraction bands.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Pore-forming proteinaceous exotoxins have been characterized for a large variety of clinically important Gram-positive and Gram-negative bacteria, including S aureus, Streptococcus pyogenes, Streptococcus pneumoniae, E coli, Proteus spp, and Pseudomonas aeruginosa.^{22 23 24 25 26 27 28} Transmembrane pore formation with subsequent electrolyte fluxes and secondary signaling events has been noted as a basic toxin mechanism. A large number of biological effects were found to be provoked by these agents; however, their impact on the coronary vasculature and cardiac performance is largely unknown.

The present study characterized staphylococcal -toxin as a potent inducer of cardiac abnormalities, including a marked coronary vasoconstrictor response and a severe depression of myocardial contractility. Clearly, these changes were caused by the staphylococcal exotoxin and not by any contamination with LPS and LPS-related cytokine generation. First, no LPS is detectable in the purified toxin preparation.²⁹ Second, sterile tubing was used throughout, and the recirculating buffer fluid was repeatedly proved to be endotoxin-free (LPS <20 pg/mL, the detection limit of the limulus-based LPS assay used). Third, administration of large quantities of LPS in the absence of -toxin did not reproduce the -toxin–elicited changes. Moreover, preapplication of LPS did not enhance the exotoxin-induced changes. These data do not exclude a role of endotoxin in eliciting cardiac abnormalities in sepsis; however, under the present experimental conditions, no such effect was demonstrated in rat hearts.

Although the primary feature of staphylococcal -toxin, its capability to induce circumscribed membrane lesions, might favor the assumption that the cardiac abnormalities are caused via overt cell damage, this is evidently not the case. Markers of cell injury (CK, LDH, K⁺) did not differ between toxin-free and toxin-perfused hearts, and microscopic studies after application of -toxin showed no signs of endothelial or myocardial cell necrosis. Instead, the present data collectively indicate that the -toxin–elicited cardiac abnormalities are largely attributable to toxin-induced thromboxane formation: (1) the formation of this vasoconstrictor agent was enhanced in toxin-treated hearts; (2) the cyclooxygenase inhibitors indomethacin and acetylsalicylic acid fully blocked thromboxane synthesis, coronary vasoconstrictor response, and depression of myocardial contractility; and (3) the same effects on heart physiology were caused by the specific thromboxane receptor antagonist daltroban, whereas thromboxane generation was unaffected by this agent. These findings are in line with previous studies of rabbit lungs, in which thromboxane-mediated vasoconstriction was noted to be the main contributor to -toxin–elicited acute pulmonary hypertension.^{30 31} The strong vasoconstrictive potency of thromboxane evidently surpasses the vasodilatory capacity of prostacyclin, which is generated with comparable kinetics but in higher quantities in response to the -toxin challenge; low numbers of prostacyclin receptors in the coronary circulation might contribute to this finding. In contrast, cysteinyl-leukotrienes, another eicosanoid species with vasoconstrictive potency, were not discovered in -toxin–challenged hearts, and the lipoxygenase inhibitor NDGA did not suppress the cardiac abnormalities. Although thromboxane generation in isolated rat hearts has been described in several studies, the source of thromboxane in the rat hearts still remains unclear. However, thromboxane generation by endothelial cells of rat aorta, in addition to rat vascular smooth muscle cells, was described.^{32 33} Moreover, endothelial cells were identified as the source of -toxin–induced prostanoid liberation.³⁴ The coronary endothelium may well be a candidate for the -toxin–induced thromboxane generation in the isolated rat heart. The mode of action by which -toxin induces synthesis of this prostanoid remains to be established. In vitro studies in different cell types, not originating from the coronary vascular bed, suggested that transmembrane calcium flux via the toxin-elicited discrete pores may be a decisive step in the induction of eicosanoid synthesis.^{34 35}

The coronary vasoconstriction in -toxin–perfused hearts was accompanied by edema formation, plateauing after 40 minutes. At first glance, this could be attributed to enhanced pressure-induced fluid filtration; however, the edema was largely unaffected by the pharmacological interventions blocking the exotoxin-elicited pressor response, thus characterizing this finding as an independent event. In endothelial monolayers, -toxin was shown to increase permeability for water and albumin by forming large intercellular gaps.³⁶ This event was clearly independent of hydrostatic pressure and was explained by direct activation of endothelial cells with subsequent cytoskeleton rearrangement. Further studies are necessary to address the question of whether the -toxin–evoked increase in endothelial permeability, as noted, for example, in rabbit lungs in response to this agent,^{30 37} is the responsible underlying event.

Our most impressive finding was that staphylococcal -toxin causes a rapid, dose-dependent decrease in myocardial contractility. Moreover, there is strong evidence that this negative inotropism is strictly related to the -toxin–induced formation of thromboxane. Time- and dose-dependence of the decrease in LVDP and dP/dt_max matched that of the toxin-induced coronary vasoconstriction very well, and all contractile abnormalities were fully blocked on cyclooxygenase inhibition and by a specific thromboxane receptor antagonist. This observation largely rules out a direct effect of -toxin on cardiomyocyte function as underlying event. Because of the constant perfusion flow, supervening the overall increase in coronary vascular resistance, global ischemia of the myocardium may also not account for the cardiodepression in the present study. In toxin-exposed rabbit lungs, the thromboxane-mediated pulmonary vasoconstriction is accompanied by a dramatic maldistribution of perfusion,³¹ and such a phenomenon, resulting in regions of oxygen deficiency next to overperfused areas, might also account for the decrease in myocardial performance in the present investigation. It is in line with this observation that all changes in cardiac physiology, coronary vasoconstriction, and depression of contractility were even further enhanced in the presence of L-NMMA, suggesting that endogenous vascular NO partly antagonizes the strong and hypothetically regionally uneven vasoconstriction by -toxin–elicited thromboxane. Such beneficial vascular effects of NO might therefore overcome putative disadvantageous effects of this agent on cardiomyocyte function, as discussed above. Although perfusion maldistribution offers an attractive explanation for the present findings, further studies are clearly necessary to verify this hypothesis.

In conclusion, purified staphylococcal -toxin exerts profound effects on rat cardiac function in the absence of circulating blood cells, plasmatic mediator systems, and endotoxin-elicited cytokine generation. Coronary vasoconstriction and depression of myocardial contractility represent the key changes, and both mediator analysis and pharmacological interventions strongly suggest that -toxin–elicited thromboxane formation is largely responsible for both events. Perfusion maldistribution in the toxin-exposed hearts offers an attractive explanation for the present findings, but this requires further elucidation. The large family of pore-forming exotoxins from Gram-positive but also from Gram-negative bacteria may thus be implicated in the loss of cardiac performance encountered in septic shock.

	Acknowledgments

 
This work was supported by the Deutsche Forschungsgemeinschaft (Si 560/4-1, Bu 819/5-1).

Received February 23, 1999; revision received June 21, 1999; accepted July 20, 1999.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Parrillo JE. Pathogenic mechanisms of septic shock. N Engl J Med. 1993;328:1471–1477.[Free Full Text]
   
2. Bone RC. The pathogenesis of sepsis. Ann Intern Med. 1991;115:457–469.
   
3. Kumar A, Parrillo JE. Clinical manifestations of cardiovascular dysfunctions in sepsis. In: Schlag G, Redl H, eds. Pathophysiology of Shock, Sepsis, and Organ Failure. Berlin, Heidelberg: Springer-Verlag; 1993:859–881.
   
4. Parker MM, McCarthy KE, Ognibene FP. Right ventricular dysfunction and dilation, similar to left ventricular changes, characterize the cardiac depression of septic shock in humans. Chest. 1990;97:126–131.[Abstract/Free Full Text]
   
5. Foulkes R, Shaw S. The cardiodepressant and vasodepressant effects of tumor necrosis factor in rat isolated atrial and aortic tissues. Br J Pharmacol. 1992;106:942–947.[Medline] [Order article via Infotrieve]
   
6. Natanson C, Eichenholz PW, Danner RL, Eichacker PQ, Hoffmann WD, Kuo GC, Banks SM, McVittie TJ, Parrillo JE. Endotoxin and tumor necrosis factor challenges in dogs simulate the cardiovascular profile of human septic shock. J Exp Med. 1989;169:823–832.[Abstract/Free Full Text]
   
7. Kumar A, Thota V, Dee L, Olson J, Uretz E, Parrillo JE. Tumor necrosis factor- and interleukin 1ß are responsible for in vitro myocardial cell depression induced by human septic shock serum. J Exp Med. 1996;183:949–958.[Abstract/Free Full Text]
   
8. Balligand JL, Ungureanu D, Kelly RA, Kobzik L, Pimental D, Michel T, Smith TW. Abnormal contractile function due to induction of nitric oxide synthesis in rat cardiac myocytes follows exposure to activated macrophage-conditioned medium. J Clin Invest. 1993;91:2314–2319.
   
9. Belder de AJ, Radomski MW, Martin JF, Moncada S. Nitric oxide and pathogenesis of heart muscle disease. Eur J Clin Invest. 1995;25:1–8.[Medline] [Order article via Infotrieve]
   
10. Avontuur JAM, Bruining HA, Ince C. Inhibition of nitric oxide synthesis causes ischemia in endotoxemic rats. Circ Res. 1995;76:418–425.[Abstract/Free Full Text]
    
11. Raper RF, Sibbald WJ, Hobson J, Neal A, Cheung H. Changes in myocardial blood flow during hyperdynamic sepsis with induced changes in arterial perfusing pressures and metabolic need. Crit Care Med. 1993;21:1192–1199.[Medline] [Order article via Infotrieve]
    
12. Hersch M, Gnidec AA, Bersten AD, Troster M, Rutledge FS, Sibbald WJ. Histologic and ultrastructural changes in nonpulmonary organs during early hyperdynamic sepsis. Surgery. 1990;107:397–410.[Medline] [Order article via Infotrieve]
    
13. Groeneveld ABJ, Van Lambalgen AA, van den Bos GC, Nauta JJP, Thijs LG. Maldistribution of heterogeneous coronary blood flow during canine endotoxin shock. Cardiovasc Res. 1991;25:80–88.[Abstract/Free Full Text]
    
14. Bloos FM, Morisaki HM, Neal AM, Martin CM, Ellis CG, Sibbald WJ. Sepsis depresses the metabolic oxygen reserve of the coronary circulation in mature sheep. Am J Respir Crit Care Med. 1996;153:1577–1584.[Abstract]
    
15. Danner RL, Suffredini AF, Natanson C, Parrillo JE. Microbial toxins: role in the pathogenesis of septic shock and multi organ failure. In: Bihari DJ, Cerra FB, eds. Multiple Organ Failure. Fullerton, Calif: Society of Critical Care Medicine; 1989:151–191.
    
16. Natanson C, Danner RL, Elin RJ, Hosseini JM, Peart KW, Banks SM, Mac Vittie TJ, Walker RI, Parrillo JE. Role of endotoxemia in cardiovascular dysfunction and mortality. J Clin Invest. 1989;83:243–251.
    
17. Harshmann S, Lefferts PL, Snapper JR. Staphylococcal alpha-toxin: a study with chronically instrumented awake sheep. Infect Immun. 1992;60:3489–3496.[Abstract/Free Full Text]
    
18. Adamo P, Taylor PB, Fackrell HB. Staphylococcal alpha toxin induced cardiac dysfunction. Can J Cardiol. 1989;5:395–400.[Medline] [Order article via Infotrieve]
    
19. Bhakdi S, Muhly M, Korom S, Schmidt G. Effects of Escherichia coli hemolysin on human monocytes. J Clin Invest. 1990;85:1746–1753.
    
20. Seeger W, Obernitz R, Thomas M, Walmrath D, Holland IB, Grimminger F, Eberspächer B, Hugo F, Suttorp N, Bhakdi S. Lung vascular injury after administration of viable hemolysin-forming Escherichia coli in isolated rabbit lungs. Am Rev Respir Dis. 1991;143:797–805.[Medline] [Order article via Infotrieve]
    
21. Kiss L, Bieniek E, Weissmann N, Schütte H, Sibelius U, Günther A, Bier J, Mayer K, Henneking K, Padberg W, Grimm H, Seeger W, Grimminger F. Simultaneous analysis of 4- and 5-series lipoxygenase and cytochrome P-450 products from different biological sources by reversed-phase high-performance liquid chromatographic technique. Anal Biochem. 1998;261:16–28.[Medline] [Order article via Infotrieve]
    
22. Bhakdi S, Traum-Jensen J. Damage to mammalian cells by proteins that form transmembrane pores. Rev Physiol Biochem Pharmacol. 1987;107:147–223.[Medline] [Order article via Infotrieve]
    
23. Bhakdi S, Traum-Jensen J. Damage to cell membranes by pore-forming bacterial cytolysins. Prog Allergy. 1988;40:1–43.[Medline] [Order article via Infotrieve]
    
24. Bhakdi S, Traum-Jensen J. Alpha-toxin of Staphylococcus aureus. Microbiol Rev. 1991;55:733–751.
    
25. Valeva A, Weisser A, Walker B, Kehoe M, Bayley H, Bhakdi S, Palmer M. Molecular architecture of a toxin pore: a 15-residue sequence lines the transmembrane channel of staphylococcal alpha-toxin. EMBO J. 1996;15:1857–1864.[Medline] [Order article via Infotrieve]
    
26. Eberspächer B, Hugo F, Pohl M, Bhakdi S. Functional similarity between the haemolysins of Escherichia coli and Morganella morganii. J Med Microbiol. 1990;33:165–170.[Abstract]
    
27. Koronakis V, Cross M, Senior B, Koronakis E, Hughes C. The secreted hemolysins of Proteus mirabilis, Proteus vulgaris, and Morganella morganii are genetically related to each other and to the alpha-hemolysin of Escherichia coli. J Bacteriol.. 1987;169:1509–1515.[Abstract/Free Full Text]
    
28. Welch RA. Identification of two different hemolysin determinants in uropathogenic Proteus isolates. Infect Immun. 1987;55:2183–2190.[Abstract/Free Full Text]
    
29. Bhakdi S, Muhly M, Korom S, Hugo F. Release of interleukin 1ß associated with potent action of staphylococcal alpha-toxin on human monocytes. Infect Immun. 1989;57:3512–3519.[Abstract/Free Full Text]
    
30. Seeger W, Bauer M, Bhakdi S. Staphylococcal -toxin elicits hypertension in isolated rabbit lungs. J Clin Invest. 1984;74:849–858.
    
31. Walmrath D, Scharmann M, König R, Pilch J, Grimminger F, Seeger W. Staphylococcal -toxin induced ventilation-perfusion mismatch in isolated blood-free perfused rabbit lungs. J Appl Physiol. 1993;74:1972–1980.[Abstract/Free Full Text]
    
32. Takayasu-Okishio M, Terashita Z, Kondo K. Endothelin-1 and platelet activating factor stimulate thromboxane biosynthesis in rat vascular smooth muscle cell. Biochem Pharmacol. 1990;40:2713–2717.[Medline] [Order article via Infotrieve]
    
33. Taddei S, Vanhoutte PM. Role of endothelium in endothelin-evoked contractions in the rat aorta. Hypertension. 1992;19:117–130.[Abstract/Free Full Text]
    
34. Grimminger F, Rose F, Sibelius U, Meinhardt M, Pötzsch B, Spriestersbach R, Bhakdi S, Suttorp N, Seeger W. Human endothelial cell activation and mediator release in response to the bacterial exotoxins Escherichia coli hemolysin and staphylococcal -toxin. J Immunol. 1997;159:1909–1916.[Abstract]
    
35. Suttorp N, Seeger W, Zucker-Reimann J, Roka L, Bhakdi S. Mechanism of leukotriene generation in polymorphonuclear leukocytes by staphylococcal alpha-toxin. Infect Immun. 1987;55:104–110.[Abstract/Free Full Text]
    
36. Suttorp N, Hessz T, Seeger W, Wilke A, Koob R, Lutz F, Drenckhahn D. Bacterial exotoxins and endothelial permeability for water and albumin in vitro. Am J Physiol. 1988;255:C368–C376.[Abstract/Free Full Text]
    
37. Seeger W, Birkemeyer G, Ermert L, Suttorp N, Bhakdi S, Duncker HR. Staphylococcal -toxin-induced vascular leakage in isolated perfused rabbit lungs. Lab Invest. 1990;63:341–349.[Medline] [Order article via Infotrieve]
    

This article has been cited by other articles:

				U. Grandel, M. Hopf, M. Buerke, K. Hattar, M. Heep, L. Fink, R. M. Bohle, S. Morath, T. Hartung, S. Pullamsetti, et al. Mechanisms of Cardiac Depression Caused by Lipoteichoic Acids From Staphylococcus aureus in Isolated Rat Hearts Circulation, August 2, 2005; 112(5): 691 - 698. [Abstract] [Full Text] [PDF]

				J.-J. Boffa, A. Just, T. M. Coffman, and W. J. Arendshorst Thromboxane Receptor Mediates Renal Vasoconstriction and Contributes to Acute Renal Failure in Endotoxemic Mice J. Am. Soc. Nephrol., September 1, 2004; 15(9): 2358 - 2365. [Abstract] [Full Text] [PDF]

				B. Lambermont, A. Ghuysen, P. Kolh, V. Tchana-Sato, P. Segers, P. Gerard, P. Morimont, D. Magis, J.-M. Dogne, B. Masereel, et al. Effects of endotoxic shock on right ventricular systolic function and mechanical efficiency Cardiovasc Res, August 1, 2003; 59(2): 412 - 418. [Abstract] [Full Text] [PDF]

				D. Pruefer, J. Makowski, M. Schnell, U. Buerke, M. Dahm, H. Oelert, U. Sibelius, U. Grandel, F. Grimminger, W. Seeger, et al. Simvastatin Inhibits Inflammatory Properties of Staphylococcus aureus{alpha}-Toxin Circulation, October 15, 2002; 106(16): 2104 - 2110. [Abstract] [Full Text] [PDF]

				U. Grandel, M. Reutemann, L. Kiss, M. Buerke, L. Fink, E. Bournelis, M. Heep, W. Seeger, F. Grimminger, and U. Sibelius Staphylococcal alpha -toxin provokes neutrophil-dependent cardiac dysfunction: role of ICAM-1 and cys-leukotrienes Am J Physiol Heart Circ Physiol, March 1, 2002; 282(3): H1157 - H1165. [Abstract] [Full Text] [PDF]

				F. Rose, G. Dahlem, B. Guthmann, F. Grimminger, U. Maus, J. Hanze, N. Duemmer, U. Grandel, W. Seeger, and H. A. Ghofrani Mediator generation and signaling events in alveolar epithelial cells attacked by S. aureusalpha -toxin Am J Physiol Lung Cell Mol Physiol, February 1, 2002; 282(2): L207 - L214. [Abstract] [Full Text] [PDF]

				U. Grandel, L. Fink, A. Blum, M. Heep, M. Buerke, H.-J. Kraemer, K. Mayer, R. M. Bohle, W. Seeger, F. Grimminger, et al. Endotoxin-Induced Myocardial Tumor Necrosis Factor-{alpha} Synthesis Depresses Contractility of Isolated Rat Hearts : Evidence for a Role of Sphingosine and Cyclooxygenase-2-Derived Thromboxane Production Circulation, November 28, 2000; 102(22): 2758 - 2764. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sibelius, U. Articles by Grimminger, F. Search for Related Content PubMed PubMed Citation Articles by Sibelius, U. Articles by Grimminger, F. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB ACETYLSALICYLIC ACID INDOMETHACIN POTASSIUM Related Collections Contractile function