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J Appl Physiol 96: 853-860, 2004. First published October 10, 2003; doi:10.1152/japplphysiol.00086.2003
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Nitric oxide-cGMP pathway is involved in endotoxin-induced contractile dysfunction in rat hearts

Tetsuya Tatsumi,1,* Natsuya Keira,1,* Kazuko Akashi,1 Miyuki Kobara,1 Satoaki Matoba,1 Jun Shiraishi,1 Satoshi Yamanaka,1 Akiko Mano,1 Mitsuo Takeda,1 Susumu Nishikawa,1 Jun Asayama,2 Henry Fliss,3 and Masao Nakagawa1

1Second Department of Medicine, Kyoto Prefectural University of Medicine, Kyoto 602-8566; 2Department of Clinical Pharmacology, Kyoto Pharmaceutical University, Kyoto 607-8414, Japan; and 3Department of Molecular and Cellular Medicine, University of Ottawa, Ottawa, Ontario, Canada K1H 8M5

Submitted 29 January 2003 ; accepted in final form 2 September 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The mechanisms by which endotoxemia causes cardiac depression have not been fully elucidated. The present study examined the involvement of nitric oxide (NO) in this pathology. Rats were infused with lipopolysaccharide (LPS) or saline, and the plasma and myocardial and (NOx) concentrations were measured before or 3, 6, and 24 h after treatment. The hearts were then immediately isolated and mounted in a Langendorff apparatus, and left ventricular developed pressure (LVDP) was determined before biochemical analysis of the myocardium. LPS injection effected the expression of inducible NO synthase (iNOS) in the myocardium, a marked increase in plasma and myocardial NOx levels, and a significant decline in LVDP compared with saline controls. The LPS-induced NO production and concomitant cardiac depression were most pronounced 6 h after LPS injection and were accompanied by a significant increase in myocardial cGMP content. Myocardial ATP levels were not significantly altered after LPS injection. Significant negative correlation was observed between LVDP and myocardial cGMP content, as well as between LVDP and plasma NOx levels. Aminoguanidine, an inhibitor of iNOS, significantly attenuated the LPS-induced NOx production and contractile dysfunction. Furthermore, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of soluble guanylate cyclase, significantly decreased myocardial cGMP content and attenuated the contractile depression, although aminoguanidine or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one was not able to completely reverse myocardial dysfunction. Our data suggest that endotoxin-induced contractile dysfunction in rat hearts is associated with NO production by myocardial iNOS and a concomitant increase in myocardial cGMP.

contractile function; septic shock; nitric oxide synthase

SEPTIC SHOCK IS CHARACTERIZED by sustained hypotension, reduced responsiveness to vasoconstrictor agents, hypoperfusion, multiple organ damage, and extremely high mortality (27). Myocardial contractility is also markedly impaired in septic shock (26). However, the mechanisms of this cardiac depression are not well understood. In vitro studies have demonstrated that cardiac myocytes isolated from endotoxin-treated animals (5) or incubated in culture medium containing endotoxin-activated macrophages (3) show depressed contractile function, which can be reversed by nitric oxide (NO) synthase (NOS) inhibitors. In addition, several proinflammatory cytokines, such as interleukin (IL)-2, IL-6, and tumor necrosis factor (TNF)-{alpha}, can produce negative inotropic effects in isolated papillary muscle preparations by NO-dependent mechanisms (10). These studies therefore suggest that myocardial dysfunction in septic shock may be associated with production of endogenous substances, such as cytokines, which are capable of causing production of endogenous NO.

In contrast to the numerous reports with isolated myocyte or muscle preparations (2, 6, 28, 33, 36), only a few studies have undertaken to investigate the role of NO in intact beating heart models of septic shock. Because the heart contains a number of cell types, such as myocytes, endothelial cells, smooth muscle cells, and fibroblasts, all of which are capable of expressing the inducible isoform of NOS (iNOS), it is important that the heart be studied as an intact organ. However, the few studies that did examine the intact heart appear to offer conflicting views on the role of NO. One example, using isolated working heart preparations from rats that had been treated with IL-1{beta} or TNF-{alpha}, demonstrated myocardial dysfunction and provided indirect evidence for NO involvement by showing that treatment with the NOS inhibitor NG-nitro-L-arginine methyl ester prevented the contractile dysfunction (32). However, the other reports concluded that NO formation is unlikely to be the sole cause of septic shock-induced myocardial dysfunction (8, 14, 16).

In view of the uncertainty regarding the role of NO in septic shock, we have attempted to bring more clarity to this issue by examining its effects in an isolated perfused rat heart model. Previous reports have suggested that NO can disrupt cellular energy balance through the inhibition of mitochondrial respiration (9, 12, 40). Furthermore, although it is well established that NO activates soluble guanylate cyclase and increases intracellular cGMP levels with a concomitant reduction in intracellular Ca2+ concentration (3, 5, 6, 28, 33), it is largely unknown whether cGMP can directly mediate myocardial dysfunction in septic shock. We therefore used direct and indirect means to examine the effect of endotoxin on myocardial function. We directly determined myocardial NO production, left ventricular developed pressure (LVDP), iNOS expression, and plasma and (NOx) concentration, as well as myocardial cGMP and ATP content. Furthermore, we indirectly assessed the role of NO and cGMP with aminoguanidine, an inhibitor of iNOS, and with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase (24).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
General experimental scheme. All test animals were handled in compliance with the guidelines for the care and use of laboratory animals of the National Institutes of Health. Salmonella typhosa LPS (15 mg/kg body wt, LPS group) or saline alone (control group) was injected intravenously into the tail vein of Sprague-Dawley rats, and systolic blood pressure and heart rate were monitored using tail cuffs. Blood samples were collected for plasma NOx determination before (0 h) or 3, 6, and 24 h after LPS or saline injection. The hearts were then rapidly isolated, mounted, and perfused on a Langendorff apparatus, and cardiac function (LVDP), as well as coronary flow, was evaluated after a 20-min equilibration period. The hearts were then rapidly chilled, and myocardial tissue was collected for biochemical determination of ATP, cGMP, and iNOS. Aminoguanidine (15 mg/kg body wt) was injected intraperitoneally 30 min before LPS injection. NG-monomethyl-L-arginine (L-NMMA, 10-4 or 10-3 M) was added to the perfusate, and the contractile parameters were determined 20 min thereafter. ODQ (10-7 or 10-6 M) was added to the perfusate of the isolated hearts after the 20-min equilibration period, and the contractile parameters were also determined 20 min thereafter.

Heart preparation. The isolated heart preparation was described by us previously (37). Briefly, adult male Sprague-Dawley rats (250-320 g) were anesthetized, heparin (1,000 IU/kg body wt) was injected intravenously, and the hearts were excised rapidly and mounted on a Langendorff apparatus. The hearts were perfused retrogradely at a constant perfusion pressure of 80 mmHg with Krebs-Henseleit buffer consisting of (mM) 118 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 1.8 CaCl2, 25 NaHCO3, and 11 glucose, pH 7.4, oxygenated with 95% O2-5% CO2 at 37°C. The atria were removed, and the hearts were paced at 270 beats/min with two pacing wires inserted into the right ventricular myocardium. A water-filled latex balloon was inserted into the left ventricle through the mitral valve for the measurement of LVDP. Left ventricular end-diastolic pressure was adjusted to 5-10 mmHg during the initial equilibration.

NOx assay. Plasma and myocardial NOx levels were determined using an autoanalyzer (model ENO-10, Eicom, Kyoto, Japan) employing an analytical column coupled to a copperized cadmium reduction column to reduce to , which was then reacted with Griess reagent to produce a product absorbing at 540 nm (13). For myocardial NOx assay, the isolated left ventricle was minced with scissors and homogenized in 4 vol of methanol and centrifuged at 10,000 g at 4°C for 10 min. The supernatant was then mixed with the same volume of the mobile phase (supplied by Eicom). and were used as reference standards.

Cytokine assay. Plasma IL-1{beta}, TNF-{alpha}, and interferon (IFN)-{gamma} content was measured 6 h after LPS or saline injection using commercially available ELISA kits for rat IL-1{beta} (BIOSOURSE), TNF-{alpha} (BIOSOURSE), and IFN-{gamma} (Genzyme) according to the manufacturers' instructions.

Western blot analysis for iNOS. At the end of the perfusion period, the hearts were chilled rapidly, and the ventricular myocardium was minced and homogenized at 0°C in phosphate-buffered saline containing 0.5 mM phenylmethylsulfonyl fluoride and centrifuged at 27,000 g for 10 min. Samples of the supernatant were subjected to standard electrophoresis and blotting techniques and probed with an iNOS-specific antibody (Transduction Laboratories, Lexington, KY; 1:2,500 dilution) at 4°C overnight and then with horseradish peroxidase-conjugated anti-mouse Ig (Amersham) for 1 h, and the bands were visualized using a chemiluminescence kit (Amersham).

Myocardial ATP and cGMP determination. Isolated hearts from 6-h control or LPS-treated rats were freeze-clamped with Wollenberger tongs. For ATP determination, frozen tissue was lyophilized overnight. Lyophilized tissue (40 mg) was then homogenized in 0.6 N perchloric acid at 0°C and centrifuged at 460 g for 10 min at 4°C. The supernatants were neutralized with KOH to pH 5.0-7.0. After 10 min, the extracts were centrifuged to remove the KClO4, and the supernatants were used for the assays. ATP content was measured by high-performance liquid chromatography (LC-9A liquid chromatograph, Shimadzu, Kyoto, Japan) and is presented as millimoles per gram dry weight (1). For cGMP determination, frozen tissue (200-300 mg) was treated with 0.25 ml of 6% trichloroacetic acid at 0°C and centrifuged at 1,000 g for 10 min. The supernatant was extracted three times with 3 ml of diethyl ether saturated with water, and the aqueous phase was collected and stored at -80°C. cGMP concentration was measured by radioimmunoassay as described previously (15). The amount of cGMP was normalized to protein content in the myocardium (20).

Statistical analysis. Data were analyzed by two-way ANOVA, with data at each time point analyzed by one-way ANOVA and Bonferroni's multiple comparison or were analyzed by one-way ANOVA and Bonferroni's multiple comparison. Values are means ± SE. P < 0.05 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Hemodynamics. Intravenous LPS injection resulted in a significant decline in blood pressure in rats within 3 h and a further decline to 64% of control by 6 h (Table 1). Heart rate increased significantly to 1.7 times that of control at 6 h. Aminoguanidine treatment significantly blocked the decline in systolic blood pressure but did not alter heart rate. There was no significant decrease in body weight throughout the protocol (data not shown).


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  		Table 1. Effect of LPS administration on systolic blood pressure and heart rate in rats

 

NOx levels and myocardial function. In contrast to control rats, in which the concentration of plasma NOx remained relatively constant, LPS injection resulted in a time-dependent increase in plasma NOx from 21.0 ± 3.2 µM immediately before injection (0 h) to a maximum level 46 times that of control by 6 h and then a decline at 24 h (Table 2). LPS injection also resulted in a similar time-dependent increase in myocardial NOx from 20.5 ± 4.3 pmol/mg protein before injection (0 h) to a maximum level 7.5 times that of control by 6 h and then a decline at 24 h (Table 2). Hearts that were isolated and mounted on a Langendorff apparatus after LPS injection showed a significant time-dependent depression in LVDP from an initial level of 106.0 ± 2.3 mmHg (0 h) to a minimum of 42% of control values in hearts isolated and mounted 6 h after LPS treatment (Table 2). LVDP subsequently increased slightly by 24 h. Coronary flow did not change significantly from initial values (14.0 ± 1.1 ml/min at 0 h) in hearts isolated from LPS-treated rats (data not shown).


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  		Table 2. Effect of LPS administration on plasma NOx concentration, myocardial NOx levels, and LVDP in isolated perfused hearts

 

Plasma cytokine levels. Cytokine concentrations were measured 0, 3, 6, and 24 h after LPS infusion (Table 3). LPS injection did not affect plasma IL-1{beta} during the experimental protocol. In contrast, LPS resulted in a time-dependent increase in TNF-{alpha} and IFN-{gamma} from 98.8 ± 21.5 pg/ml and 0.5 ± 0.2 ng/ml, respectively, before injection (0 h) to a near-maximum level 4.4 and 50.2 times, respectively, that of control by 6 h and then a decline at 24 h. Aminoguanidine treatment did not significantly inhibit the LPS-induced increase in plasma cytokine levels.


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  		Table 3. Effect of LPS administration on plasma concentrations of IL-1{beta}, TNF-{alpha}, and IFN-{gamma}

 

Effect of aminoguanidine. The effects of LPS and aminoguanidine on LVDP, myocardial NOx levels, and myocardial ATP content 6 h after LPS administration are shown in Fig. 1. Aminoguanidine alone did not significantly affect LVDP or myocardial NOx content. However, it significantly attenuated the LPS-induced contractile dysfunction and the increase in myocardial NOx levels. Aminoguanidine also markedly inhibited the increase in plasma NOx 6 h after LPS injection (data not shown). Myocardial ATP content was 17.8 ± 0.9 mmol/g dry wt in control hearts, and there was no significant reduction in ATP content 6 h after LPS administration in the presence or absence of aminoguanidine.



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  		Fig. 1. Effect of aminoguanidine (AG) on left ventricular developed pressure (LVDP; A), myocardial and (NOx; B) levels, and myocardial ATP content (C). Aminoguanidine was injected intraperitoneally 30 min before saline (Cont) or lipopolysaccharide (LPS) treatment, and LVDP, myocardial NOx, and myocardial ATP content were measured 6 h after LPS injection. Values are means ± SE (n = 6). *P < 0.01 vs. Cont; {dagger}P < 0.01 vs. LPS by 1-way ANOVA and Bonferroni's multiple comparison.

 

Expression of myocardial iNOS. Figure 2 illustrates a typical iNOS immunoblot obtained with myocardial extracts from a control heart and hearts isolated 6 h after LPS injection with or without prior treatment with aminoguanidine. In contrast to the total absence of iNOS immunoreactivity in the control hearts, LPS significantly increased iNOS expression. Aminoguanidine treatment attenuated LPS-induced iNOS expression.



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  		Fig. 2. Expression of inducible nitric oxide synthase (iNOS) in LPS-treated hearts. Cytosolic fractions were obtained from hearts isolated 6 h after saline (Cont) or LPS injection in the presence or absence of aminoguanidine, and aliquots containing 100 µg of protein were subjected to Western blot analysis. Immunoblots are representative of >=3 similar results.

 

Effect of L-NMMA. Hearts isolated 6 h after LPS injection were mounted on a Langendorff apparatus and, after an initial equilibration period of 20 min, were treated with 10-4 or 10-3 M L-NMMA. The effects on LVDP, coronary flow, and myocardial cGMP content were measured before and 20 min after administration of L-NMMA. L-NMMA significantly decreased LVDP and coronary flow in control hearts (Fig. 3). In contrast, L-NMMA did not alter LVDP in the LPS-treated hearts, although it significantly decreased coronary flow. LPS induced a significant increase in myocardial cGMP content to 1.58 times control. Moreover, L-NMMA did not significantly affect myocardial cGMP content in control and LPS-treated hearts, although it tended to decrease cGMP content in LPS-treated hearts.



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  		Fig. 3. Effect of 10-4 or 10-3 M NG-monomethyl-L-arginine (L-NMMA) on LVDP (A), coronary flow (CF; B), and myocardial cGMP (C) in hearts that were isolated 6 h after injection of saline (Cont) or LPS and perfused in a Langendorff apparatus. Open bars, before L-NMMA; solid bars, after L-NMMA. Values are means ± SE (n = 5). {ddagger}P < 0.05; §P < 0.01 vs. before treatment; {dagger}P < 0.01 vs. Cont + L-NMMA(-) by 1-way ANOVA and Bonferroni's multiple comparison.

 

Effect of soluble guanylate cyclase inhibitor. Hearts isolated 6 h after LPS injection were mounted on a Langendorff apparatus and, after an initial equilibration period of 20 min, were treated with 10-7 or 10-6 M ODQ, an inhibitor of soluble guanylate cyclase. The effects of ODQ on LVDP, coronary flow, and myocardial cGMP content were similarly measured before and 20 min after administration of ODQ (Fig. 4). In control hearts, ODQ significantly decreased LVDP and coronary flow. In contrast, ODQ decreased coronary flow in LPS-treated hearts and partially but significantly improved the LPS-induced depression of LVDP. ODQ at 10-6 M significantly decreased myocardial cGMP content in control hearts. Moreover, it significantly reversed the LPS-induced increase in myocardial cGMP content to the control level.



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  		Fig. 4. Effect of 10-7 or 10-6 M 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) on LVDP (A), coronary flow (B), and myocardial cGMP (C) in hearts that were isolated 6 h after injection of saline (Cont) or LPS and perfused in a Langendorff apparatus. Open bars, before ODQ; solid bars, after ODQ. Values are means ± SE (n = 5). {ddagger}P < 0.05; §P < 0.01 vs. before treatment; ¶P < 0.01 vs. Cont + ODQ(-); #P < 0.01 vs. LPS + ODQ(-) by 1-way ANOVA and Bonferroni's multiple comparison.

 

Correlation between NO or cGMP content and myocardial function. There was a statistically significant negative correlation between LVDP and cGMP content (r = 0.635, P < 0.001) as well as between LVDP and plasma NOx concentration (r = 0.621, P < 0.01; Fig. 5).



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  		Fig. 5. Relation between LVDP (A) and plasma NOx concentration (A) or myocardial cGMP content (B) before or after LPS injection. A: y = 83.8 - 0.043x (r = 0.621, P < 0.01, n = 24). {triangleup}, LPS 0 h; {bullet}, LPS 3 h; {square}, LPS 6 h; {blacksquare}, LPS 24 h. B: y = 150.4 - 11.7x (r = 0.635, P < 0.001, n = 30). {circ}, Cont; {square}, Cont + 10-7 M ODQ; {triangleup}, Cont + 10-6 M ODQ; {bullet}, LPS; {blacksquare}, LPS + 10-7 M ODQ; {blacktriangleup}, LPS + 10-6 M ODQ.

 


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Using a combination of an in vivo rat model and an associated isolated beating heart model, our study clearly shows that septic shock caused an increased production of cytokines, NO, and cGMP, which was associated with a sharp decline in myocardial function. Specifically, our study directly demonstrates that the intravenous injection of LPS into rats caused a sharp increase in plasma and myocardial NO, which was associated with a rapid upregulation of myocardial iNOS and an increase in myocardial cGMP. The increased levels of NO and cGMP were significantly and independently correlated with a decline in LVDP in hearts isolated from the treated rats. Our study also provided strong indirect evidence for this correlation by showing that pretreatment of the rats with aminoguanidine, an iNOS blocker, attenuated the expression of the myocardial iNOS, lowered the level of plasma and myocardial NO, and significantly inhibited the LPS-induced contractile dysfunction. Furthermore, an inhibitor of soluble guanylate cyclase, ODQ, decreased myocardial cGMP content and partially but significantly attenuated the myocardial contractile dysfunction, suggesting that NO and cGMP may contribute to the contractile dysfunction.

In the present study, the plasma concentrations of TNF-{alpha} and INF-{gamma}, cytokines that are capable of inducing the expression of iNOS (12, 21), were significantly increased at 3 h and reached a near-maximum level 6 h after the administration of LPS, concomitantly with the maximal upregulation of myocardial iNOS. These data therefore suggest that LPS increased cytokine production, which, in turn, increased myocardial iNOS expression. Because endotoxin can upregulate endothelial NOS as well as iNOS, it is possible that in our model the elevated plasma NO was the product of both enzymes (30, 31, 35). However, because aminoguanidine is demonstrated to prevent the expression of iNOS (29) and is ~30 times more effective at inhibiting iNOS than endothelial NOS, it is likely that NO was produced primarily by iNOS. Recent reports have also demonstrated that the effect of LPS on iNOS is not tissue specific, producing an increase in iNOS mRNA expression in numerous organs such as the lungs, liver, spleen, skeletal muscle, kidney, and heart of rats and resulting in a dramatic elevation in circulating NO levels (7, 19, 21). The large increase in plasma NO that was observed in our study was therefore probably produced by a number of tissues in addition to the myocardium.

Recent reports have shown that the production of excessive amounts of NO by iNOS can inhibit mitochondrial respiration and cause deleterious disturbances in the energy balance in a variety of cells (9, 12), including cardiac myocytes (40). We also recently demonstrated that excessive NO production in cultured rat neonatal cardiac myocytes exposed to the proinflammatory cytokine IL-1{beta} for 48 h can lower myocardial energy production through the inhibition of the mitochondrial iron sulfur enzymes (38). It was therefore surprising that, in the present study, myocardial ATP content was not significantly decreased 6 h after LPS injection, despite the pronounced LPS-induced cardiac depression. Our data, therefore, indicate that the deleterious effects of NO in our model were not mediated through inhibition of cardiac energy production, suggesting that other pathways may be involved. One of these pathways may involve the production of cGMP.

In the present study, inhibition of NOS or soluble guanylate cyclase significantly decreased LVDP and coronary flow in control hearts. It is likely that the depression of cardiac contractility is derived from the reduced coronary flow. In addition, Kojda et al. (18) recently showed that inhibitors of NOS or soluble guanylate cyclase attenuate the contractile function in isolated normal rat hearts. They suggested that endogenous NO production exerts a positive inotropic effect that is mediated by production of cGMP, because mildly increased cGMP can inhibit cAMP hydrolysis by an inhibition of phosphodiesterase. Thus increased cAMP could be involved in the increased contractile response via a stimulation of cAMP-dependent protein kinase in control hearts. In contrast, our study shows that inhibition of NOS did not affect LVDP in LPS-treated hearts, although it significantly decreased coronary flow, consistent with the previous report (22). Moreover, significant increases in myocardial cGMP levels were observed 6 h after LPS injection, correlating with the period of maximal cardiac dysfunction. The ability of ODQ to block cGMP production in our studies and to concomitantly attenuate cardiac dysfunction strongly suggests that cGMP played a significant role in LPS-mediated cardiac injury, although aminoguanidine or ODQ was not able to completely reverse myocardial dysfunction. These data therefore suggest that, in LPS-treated hearts, cGMP may initiate injurious processes that are distinct from those in normal hearts.

Our surprising findings with the NOS inhibitor L-NMMA suggest that cGMP may actually play a more direct role than NO in the etiology of septic shock-mediated cardiac dysfunction. We show that the addition of a bolus of L-NMMA to the perfusate of the isolated hearts did not improve LVDP or decrease cGMP content, indicating that the in vitro production of cardiac NO is not acutely or directly responsible for the myocardial dysfunction but may be indirectly involved, possibly as a result of cGMP production. The similar inability of an NOS inhibitor to restore cardiac contractility after LPS treatment in another in vitro study also argues in favor of a more indirect role for NO in this pathology (8, 23). It is therefore tempting to speculate that LPS-associated cardiac dysfunction may be more directly dependent on myocardial cGMP content than on local NO levels.

The precise mechanism by which an elevation in myocardial cGMP may cause cardiac dysfunction is not well understood, and the available experimental data are conflicting. For example, in studying the involvement of cellular Ca2+, a number of previous studies have shown that cGMP can decrease myocardial cytoplasmic Ca2+ concentration in vitro (3, 6, 28, 33) and modulate myocardial contractility and relaxation by lowering myofilament response to Ca2+ (33). However, in guinea pig ventricular myocytes, cGMP was found to increase sarcolemmal Ca2+ influx by inhibiting a phosphodiesterase (25), whereas in amphibian and avian myocytes it reduces the sarcolemmal L-type Ca2+ current by activating phosphodiesterases (11, 39). Furthermore, in rat ventricular myocytes, cGMP predominantly inhibited this Ca2+ current by a mechanism involving cGMP-dependent protein kinase (34). In view of this apparent multiplicity of effects, all that can be safely proposed at this time is that myocardial cGMP appears to contribute significantly to the cardiac dysfunction associated with septic shock. Moreover, recent observations have proposed that an endogenously produced small increase in cGMP elicits a positive inotropic effect, presumably mediated by a cAMP-dependent pathway, but a high increase in cGMP elicits a depression of contractility mediated by activation of cGMP-dependent protein kinase in isolated rat ventricular myocytes (17). Our data obtained from control and LPS-treated hearts, therefore, might reflect a biphasic manner of NO-cGMP dependent contractile force.

In the present study, aminoguanidine did not completely reverse the LPS-induced contractile dysfunction. In addition, marked hemodynamic changes, such as a fall in blood pressure and an increase in heart rate, were also seen after LPS injection. These data, therefore, suggest the involvement of other humoral and hemodynamic factors, in addition to NO, in this injurious process. For example, the marked elevation in plasma TNF-{alpha} may directly suppress cardiac contractility (4). Our studies have also detected a limited sequestration of mononuclear and polymorphonuclear leukocytes in the LPS-treated myocardial tissue (data not shown), raising the possibility of a contribution by inflammatory cells to this process.

In summary, the present study demonstrates that cardiac dysfunction induced by LPS is partially mediated by the NO-cGMP pathway, with cGMP likely providing a more direct injurious effect. However, the involvement of other humoral factors cannot be excluded. The principal contribution of this study is that it provides direct and indirect data illustrating this relation in vivo, as well as in an associated isolated rat heart model of LPS injury.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. Tatsumi, Second Dept. of Medicine, Kyoto Prefectural Univ. of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan (E-mail: tatsumi{at}koto.kpu-m.ac.jp).

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.

* T. Tatsumi and N. Keira contributed equally to this work. Back


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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