INTRODUCTION

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When cardiovascular dysfunction complicates sepsis, the mortality rate approximately doubles (1, 2), contributing to the high and rising incidence of death due to serious infections. An important aspect of cardiovascular dysfunction of sepsis is decreased left ventricular function (3). A number of components of the septic inflammatory cascade have been shown to contribute to decreased ventricular function, including tumor necrosis factor (TNF) (4, 5), other proinflammatory cytokines (6), and leukocytes (7). TNF and other proinflammatory cytokines may mediate part of their effect via nitric oxide (NO), either via NO's effect of myocyte cyclic guanosine 3',5'-monophosphate (cyclic GMP) or via peroxynitrite formed by combination of NO with oxygen radicals (8). Leukocytes also mediate some of their damaging effects in other tissues via oxygen radical formation (9). Whether oxygen radicals generated by myocytes, leukocytes (10, 11), or other cells, contribute to ventricular dysfunction during sepsis is not known. However, release of reactive oxygen radicals by leukocytes is an important contributor to the pathogenesis of ischemia-reperfusion injury in the heart (12). Thus, it is reasonable to postulate that oxygen radicals may be important in causing ventricular dysfunction in sepsis.

Glutathione (GSH) is an important endogenous antioxidant that protects cells and tissues against oxygen radical damage (15). Continued release of oxygen radicals during sepsis depletes the supply of glutathione, leaving tissues vulnerable to damage by oxygen radicals (9). Restoring glutathione concentrations is necessary to continue its protective role, but increasing glutathione levels can be problematic. Direct administration of glutathione is restricted because glutathione is easily oxidized and hydrolyzed by intestinal and hepatic -glutamyltransferase (18). The rate of glutathione synthesis is usually limited by the amount of cysteine present (15). Cysteine, a glutathione precursor, is rapidly oxidized, has limited cell uptake, and may be toxic when present extracellularly at high concentrations (19, 20). However, glutathione repletion can be achieved effectively by administering L-2-oxothiazolidine-4-carboxylic acid, OTZ (Procysteine; Transcend Therapeutics, Inc., Cambridge, MA) (21). This compound is converted to cysteine by the intracellular enzyme, oxoprolinase (22).

Accordingly, we asked if OTZ could prevent decreased ventricular contractility observed after endotoxin infusion (5, 23). We reasoned that OTZ would replenish depleted glutathione stores and enable the glutathione cycle to catabolize oxygen radicals. To address our hypothesis we used an isolated heart perfused by a support rabbit (7, 23). Endotoxin infusion into the support rabbit provided a whole animal model of sepsis while the isolated heart allowed us to control coronary perfusion pressure, preload, afterload, heart rate, and eliminate mechanical interaction of the heart with surrounding structures.

	METHODS

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This study was approved by the University of British Columbia Animal Care Committee and adheres to the Canadian guidelines on animal experimentation.

Pretreatment Protocol

Fifty-six rabbits (New Zealand White) in the experiments were pretreated with either a total of 2.4 g/kg of a 10% OTZ solution (OTZ groups), or an equal volume and osmolality of saline (Saline groups), administered in three divided doses subcutaneously at 4-h intervals beginning 24 h prior to the experiment.

Surgical Preparation of the Support Rabbit

Twenty-eight of the above rabbits (3.5 ± 0.5 kg) were anesthetized using -chloralose (Sigma, St. Louis, MO) 55 mg/kg and urethane (32%; Sigma) 4 ml/kg intravenously. A tracheotomy was performed and the rabbits were ventilated with room air and supplemental oxygen to maintain PCO₂ between 30 to 40 mm Hg and PO₂ above 100 mm Hg. Polyethylene catheters (interior diameter [i.d.] 1.67 mm, outer diameter [o.d.] 2.42 mm; Intramedic, Becton Dickinson, Parsippany, NJ) were inserted into the right carotid artery and the left external jugular vein. A third polyethylene catheter (i.d. 1.14 mm, o.d. 1.57 mm) was inserted into the left femoral artery to monitor arterial blood pressure. Rabbits were anticoagulated with 1,000 IU/kg heparin (Organon; Teknika, Toronto, ON, Canada) intravenously. A crystalloid solution (Plasma-Lyte A, with 7 IU/ml heparin; Baxter Corp., Toronto, ON, Canada) was continuously infused via the left external jugular vein catheter. Generally, the animals required 20 to 30 ml crystalloid during the surgical preparation of the isolated heart, and an additional 30 to 40 ml/h throughout the experimental period. When pH fell below 7.35, 5-ml boluses of 6% sodium bicarbonate were given (approximately 20 to 30 ml/experiment). A rectal temperature probe was inserted and the core body temperature of the animal was maintained at 38.9 ± 0.2° C using a heating blanket.

Langendorff Column and Extracorporeal Circuit

The support rabbit was then connected to a circuit which perfused a Langendorff column. Arterial blood from the right carotid catheter of the support rabbit was pumped via a roller pump to an open perfusion column connected to a heated heart chamber, to produce a coronary perfusion pressure of 75 mm Hg at the level of the aortic valve of the isolated heart. Blood overflowing from the perfusion column and venous blood from the isolated heart was pumped back via a second roller pump through a 40-µm blood filter (SQ40S Blood Transfusion filter; Pall Biomedical Products Corporation, East Hills, NY) to the support animal via the external jugular vein catheter. The total volume of this circuit was approximately 35 ml.

Surgical Preparation of the Isolated Heart

The remaining 28 (out of the initial 56) rabbits (2.5 ± 0.5 kg) were anesthetized with a mixture of ketamine 80 mg/kg and xylazine 5 mg/ kg subcutaneously. A midline sternotomy and pericardiotomy was performed. The hearts were rapidly excised and affixed via the aorta to the Langendorff column. The pulmonary artery was incised at the base of the right ventricular outflow tract to allow venous drainage from the right ventricle. A 5-mm incision was made in the left atrium and a 7-French single-lumen pressure transducer (Millar Instruments Inc., Houston, TX) surrounded by a saline-filled latex balloon (unstressed volume 3 ml) was inserted into the left ventricle and secured using an external ligature surrounding the left atrium. Pacing electrodes were attached to both the left and right atria and the hearts were paced at 150 beats/min. The ventricular balloon was inflated with normal saline via the Millar catheter lumen until a left ventricular end-diastolic pressure of 4 mm Hg was achieved (volume approximately 200 µl). After the ventricular balloon and pacing electrodes were in place, the isolated heart was then allowed to beat isovolumically for 15 min.

Measurement of Left Ventricular Function

Ventricular contractility was measured using the slope of the end-systolic pressure-volume relationship, Emax (24). To calculate Emax, the intraventricular balloon was inflated using a syringe pump at a constant rate of 800 µl/min to a maximal volume determined as the volume at which cardiac dysrhythmia occurred (approximately 400 µl). A constant balloon inflation rate allowed time measurements to be converted directly to intraventricular volume measurements. The maximal balloon inflation volume never approached the balloon's unstressed volume of 3 ml. During inflation, left ventricular pressure was sampled at 100 Hz and stored in digital format. The slope of the best-fit line to the ascending ramp of peak systolic pressures is Emax. Immediately after inflation to maximal volume, the balloon was deflated to the initial volume of approximately 200 µl.

Measurements of Perfusing Blood

Arterial PO₂, PCO₂, and pH were measured using a blood gas analyzer (ABL30 Radiometer, Copenhagen, Denmark). We also measured arterial lactate concentration (YSI 2300 Stat Glucose-Lactate analyzer; YSI Incorporated, Yellow Springs, OH) and hemoglobin (IL482 Co-oximeter; Instrumentation Laboratory, Lexington, MA).

Serum TNF Bioassay

Serum TNF concentrations were measured using the WEHI bioassay (25). Briefly, 5 × 10⁵ WEHI 164 subclone 13 cells in 100 µl were added to 100 µl volumes of serial dilutions of serum samples, and were incubated overnight. Then cell viability was measured with a colorimetric assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma). During the final 6 h of the incubations, 20 µl MTT (5 mg/ml in phosphate-buffered saline) was added to each sample. Supernatant (150 µl) was aspirated from each well and 100 µl acidified isopropanol was added. Absorbance was measured at 550 nm. TNF concentrations in experimental samples were calculated using a standard curve generated by serial dilutions of recombinant murine TNF-. These bioassays consistently detected TNF concentrations above 1 pg/ml.

Myocardial Leukocyte Content

Immediately after the conclusion of the experiment the blood perfusion circuit was interrupted and normal saline was simultaneously infused into the isolated heart to flush out red blood cells from the myocardial circulation (approximately 5 min). Glutaraldehyde 2.5% in phosphate buffer was then added to the saline perfusion circuit and the heart was perfusion fixed for 10 min. The isolated heart was then removed from the apparatus and transferred to a container of the same fixative. Left ventricular tissue sections (5 mm thickness) from hearts fixed in glutaraldehyde were dehydrated and embedded in paraffin. Serial sections (4 µm thickness) were stained with hematoxylin and eosin. The number of random fields, at ×400 original magnification, necessary to count 100 leukocytes in each group was recorded. The average number of leukocytes per field was then determined as a quantitative assessment of myocardial leukocyte content.

Experimental Protocol

On the day of the experiment an additional 2.4 g/kg OTZ 10% solution (OTZ groups) or equivalent volume of saline (Saline groups) was given intravenously at a rate of 2 ml/min over approximately 40 min (21). One hour after the start of OTZ (or saline) infusion, baseline measurements were taken and 1 mg/kg endotoxin (lipopolysaccharide; Sigma) (Endotoxin groups) or an equivalent volume of vehicle (Control groups) was given intravenously over 30 min. These two interventions defined four experimental groups which were Saline/Endotoxin (n = 7) and OTZ/Endotoxin (n = 7) to test the hypothesis that OTZ prevents endotoxin-induced ventricular dysfunction, and Saline/Control (n = 7) and OTZ/Control (n = 7) to control for time effects and for any independent effects of OTZ. Variables were measured at baseline and 6 h after the start of endotoxin infusion.

Glutathione Assay

In entirely separate experiments from those described previously, rabbits were pretreated with OTZ (n = 3) or with saline (n = 3) and endotoxin was infused, as described. Six hours after the start of endotoxin infusion, hearts were rapidly excised and snap-frozen in liquid nitrogen. Glutathione levels were measured in frozen heart tissue using the Bioxytech GSH-400 colorimetric assay (R&D Systems, Minneapolis, MN). Briefly, 500 mg of tissue were homogenized in 10 ml ice-cold 5% metaphosphoric acid (Sigma). A volume of 100 µl of supernatant was then mixed with 800 µl buffer (200 mM potassium phosphate, pH 7.8 at 25° C, containing 0.2 mM diethylenetriamine pentaacetic acid and 0.025% lubrol), 50 µl each of a solution of 1.2 × 10² M chromogenic reagent in 0.2N hydrochloric acid and 30% sodium hydroxide (Bioxytech GSH-400 kit; R&D Systems). After a 10-min incubation at 25° C, absorbance was measured at 400 nm. Glutathione levels were calculated using a standard curve generated by serial dilutions of glutathione in solution (Sigma).

Data Analysis

We tested the principal null hypothesis that there was no difference in contractility (Emax) between the four experimental groups at 6 h after the start of endotoxin infusion with a two-way repeated measures analysis of variance (ANOVA) using p < 0.05 as significant. Specifically, when we analyzed the data we included a factor for absence/ presence of OTZ, one for absence/presence of endotoxin, and an interaction term between the two. When a significant difference was identified we used a sequentially rejective Bonferroni test procedure to identify specific differences. Similar analysis was used to test for differences in other measured variables. Data are reported as mean ± standard error.

	RESULTS

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Emax decreased by 16.1 ± 4.4% in the Saline/Endotoxin group (Figure 1) but did not decrease in the OTZ/Endotoxin group (+6.3 ± 4.1%) by 6 h after the start of endotoxin infusion. Emax did not change significantly from baseline to 6 h in either of the Saline/Control or OTZ/Control groups (Figure 1). The statistical interaction between OTZ and endotoxin was significant (p = 0.029) and the Saline/Endotoxin group differed from all three other groups after correction for multiple statistical comparisons (Saline/Endotoxin versus OTZ/Endotoxin p = 0.009, Saline/Endotoxin versus Saline/Control p = 0.034, or Saline/Endotoxin versus OTZ/Control p = 0.005).

View larger version (14K): [in this window] [in a new window]   Figure 1.   Contractility, as measured by average Emax, is shown at 6 h after the start of endotoxin infusion, with standard error bars. Emax decreases significantly in the Control/Endotoxin group compared with all other groups (*p < 0.05, ANOVA). For the key prospectively planned comparison of the Control/Endotoxin to OTZ/ Endotoxin groups, p = 0.007 by ANOVA.

Serum TNF concentrations increased to the same extent in the Saline/Endotoxin and OTZ/Endotoxin groups (Figure 2). In the control groups that did not receive endotoxin, TNF was initially slightly elevated, probably related to the surgical preparation, and then decreased (Figure 2). Myocardial leukocyte content of 1.5 ± 0.2 leukocytes/field in the Saline/Control and OTZ/Control groups increased to 2.4 ± 0.2 leukocytes/field in the Saline/Endotoxin and OTZ/Endotoxin groups (p < 0.005) (Figure 3). However, OTZ administration had no significant effect on myocardial leukocyte concentration.

View larger version (16K): [in this window] [in a new window]   Figure 2.   Average serum TNF concentrations are shown with standard error bars. Endotoxin infusion causes a marked increase in serum TNF concentration (endotoxin effect, p = 0.018) but there is no statistically significant effect of OTZ (p = 0.788) and no statistically significant interaction between OTZ and endotoxin (p = 0.402). Standard errors in the Saline/Control group are so small the error bars lie within the symbols.

View larger version (41K): [in this window] [in a new window]   Figure 3.   Average leukocyte count in the myocardium (sampled immediately after the 6-h time point measurements) per ×400 field is shown for all four groups with standard error bars. ANOVA shows that there is a significant endotoxin effect (*p < 0.05) but that there is no OTZ effect (p = NS).

In separate experiments we found that myocardial glutathione concentrations were greater in rabbits treated with OTZ (104 ± 4 ng/g) than in those treated with saline (80 ± 3 ng/g) (p < 0.05) 6 h after the start of the endotoxin infusion.

There were no differences between groups in blood gas measurements, lactate, or hemoglobin of support rabbit blood that perfused the isolated hearts that could account for the measured difference in contractility between the Saline/Endotoxin and OTZ/Endotoxin groups (Table 1).

	DISCUSSION

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These results show that OTZ, a cysteine prodrug and glutathione repleting agent (16, 21), prevents the early decrease in ventricular contractility in isolated-perfused hearts following endotoxin infusion into support rabbits. Similar to previous reports (16, 17, 21, 22), OTZ administration also increased myocardial glutathione concentrations after endotoxin infusion. These results support the hypothesis that oxygen radicals contribute to the early decrease in ventricular contractility in sepsis.

OTZ administration did not alter serum TNF expression or the myocardial leukocyte retention that occurred after endotoxin infusion. One possible explanation is that TNF and oxygen radical effects are unrelated and caused by separate mechanisms. Alternatively, these data suggest that oxygen free radicals, modulated by OTZ, have myocardial depressant effects downstream in the septic inflammatory response from proinflammatory cytokines (6) and downstream from leukocytes, which accumulate in the heart (7, 10, 11, 23) and contribute to the early decrease in ventricular contractility in models of sepsis (7).

Myocardial dysfunction has been reported in human sepsis (3) and in animal models of sepsis (4, 5, 7, 10, 23). A number of mechanisms have been identified (26). Circulating myocardial depressant factors appear to contribute (26). Proinflammatory cytokines (6), notably TNF (4, 5, 6, 27), may act as myocardial depressant factors. These cytokines may mediate part of their effect via NO generation from nearby endothelium, from cardiac myocytes (8), and from other cellular sources. NO may mediate its myocardial depressant effects in several ways, including by increasing cyclic GMP activity (28) and also by forming peroxynitrite after combining with oxygen radicals (29). The importance of NO is uncertain as other studies do not show NO-mediated myocardial depression in animal models of sepsis (30). In addition, other inflammatory mediators (e.g., platelet activating factor [31] ) and leukocytes (7) appear to contribute to myocardial dysfunction in sepsis. The role that oxygen radicals play in septic myocardial dysfunction has not been determined.

In 1981, Williamson and Meister described OTZ as a cysteine delivery system and glutathione enhancer (16). Oxoprolinase, an enzyme present in most mammalian cells, metabolizes OTZ into cysteine and CO₂ at the expense of ATP. OTZ treatment maintained cellular glutathione levels when glutathione-depleting agents were given to healthy animals (32, 33) and OTZ repleted tissue glutathione levels in acutely septic rats (34). In accord with these studies we found that OTZ increased myocardial glutathione concentration, measured 6 h after endotoxin infusion. Thus, OTZ administration is an intervention that allowed us to test for a possible myocardial depressant effect of oxygen radicals.

Oxygen free radicals come from several sources and could have several effects in the heart. Activated neutrophils are important sources of oxygen radicals and activated neutrophils accumulate in the heart after endotoxin infusion in animal models (7, 10, 11, 23). Filtering leukocytes from coronary blood prevents the early decrease in ventricular contractility after endotoxin infusion in rabbits (7). Because OTZ also prevented the early decrease in contractility in a similar experimental model, it is reasonable to postulate that neutrophils mediate much of their myocardial depressant effects via oxygen radicals. However, the decrease in contractility is not necessarily due to leukocyte release of oxygen radicals. It is also possible that leukocytes decrease ventricular contractility by other means, such as cytokine release, and it is conceivable that OTZ could have protected cells from other oxidant stress generated within the myocytes. Alternatively, oxygen radicals may decrease contractility via intracellular oxidant signaling (35) without necessarily damaging tissue (36). OTZ could modify intracellular oxidant signaling (35) and prevent this effect without altering tissue morphometry.

A number of issues related to the experimental model should be addressed. The decrease in contractility and increase in serum TNF concentration observed after endotoxin administration in this model resembles that observed after acute endotoxin or proinflammatory cytokine administration in other animal models (4, 5, 7, 10, 23) and in acute studies in humans (37). However, whether this endotoxin infusion model in rabbits models the cardiovascular changes over days described in human septic shock (3) is not known. Therefore, these results should be interpreted in this light and limited to the early decrease in contractility after an initiating septic stimulus. Differences in contractility between the Saline/Endotoxin group and the OTZ/Endotoxin group were independent of any changes in myocardial perfusion pressure, preload, afterload, heart rate, and ventricular interaction (4, 24) because these factors were all kept constant in the isolated-supported heart where Emax was measured. Similarly, PO₂, PCO₂, pH, lactate, and hemoglobin of support rabbit blood that perfused the isolated hearts did not account for the measured difference in contractility between the Saline/Endotoxin and OTZ/Endotoxin groups. Although lactate rose with time in support rabbit blood, there was no significant difference between groups. Thus, the effect of OTZ treatment on endotoxin-induced ventricular dysfunction is not accounted for by these differences in perfusing blood.

In summary, OTZ treatment increases myocardial glutathione concentration and prevents the early decrease in contractility after endotoxin infusion, implying that oxygen radicals are in part responsible for impaired ventricular function. Oxygen radical-mediated ventricular dysfunction may account for some of the previously observed leukocyte-mediated myocardial dysfunction after endotoxin infusion (7).