Cardiovascular dysfunction is a major contributor in septic shock–induced mortality (1). Septic shock is characterized by both an alteration in vascular tone (2) and also by a systolic and diastolic biventricular dysfunction (3). In clinical practice, volume-resuscitated patients exhibit high cardiac output (CO) and low systemic resistance with myocardial depression despite the high CO. The overall incidence of global left ventricular (LV) hypokinesia in septic shock patients without any prior cardiac history is 60%, a value much higher than previously described (4). In 10% to 20% of septic shock patients, septic cardiomyopathy mimics a cardiogenic shock state with impaired CO leading to death (5). Several hypotheses have been proposed to explain sepsis-induced cardiac dysfunction, including the infiltration of activated leukocytes (6), and the effects of cytokines such as tumor necrosis factor α and interleukin 1β, identified as “circulating myocardial depressant factors” (7). Most of the deleterious effects of proinflammatory cytokine have been attributed to the overproduction of nitric oxide (NO), which may exert detrimental effects on the myocardium (8). The inhibition of NO synthase activity has been shown to prevent endotoxin-induced myocardial dysfunction (9). Conversely, under normal levels, NO is necessary for normal cardiac physiology and plays a protective role in the ischemic heart through numerous mechanisms (10, 11). Recent studies have highlighted the negative inotropic role of peroxynitrite (12, 13). Indeed, the direct toxicity of NO is modest but is greatly enhanced by reacting with superoxide (O2 −) to form peroxynitrite (ONOO−) (14).
Corticosteroids are recommended as an adjuvant therapy for adult septic shock patients unresponsive to fluid resuscitation and vasopressors (15). Although their intracellular mechanisms of action are not completely elucidated, it has been shown that they involve genomic and nongenomic mechanisms (16). Genomic mechanisms lead to inhibition of the nuclear factor κB pathway, resulting in reduced expression of inducible NO synthase (iNOS) (17). Over the past decade, a better understanding of sepsis pathophysiology has emerged focused on the tight coupling of inflammation and coagulation processes (18). Activated protein C (APC), a systemic endogenous anticoagulant and anti-inflammatory factor, has also been considered in the management of septic patients. Despite the disappointing results of the PROWESS SHOCK study (19), many large-scale clinical studies found beneficial effects of APC use during sepsis (20–22). Anticoagulant properties do not appear to be the most important cause of this benefit. Activated protein C also demonstrates cytoprotective, antiapoptotic, and anti-inflammatory properties that are probably at the origin of its beneficial effects (23). Lastly, APC has been demonstrated to inhibit nuclear factor κB, tumor necrosis factor α (24), or iNOS (25) pathways.
Corticosteroids and APC improve vascular function in sepsis experimental models, but data are scarce regarding their effects on myocardial function during septic shock. In a previous study from our group (26), we have observed an improvement in LV ejection fraction evaluated by echocardiography with APC after lipopolysaccharide challenge in rats. Nevertheless, because of important dependency to load conditions, ejection fraction cannot reliably evaluate intrinsic contractility, particularly in the context of low blood pressure. We therefore decided to evaluate contractile cardiac function by load-independent parameters using conductance catheter, to discriminate vascular effects—extensively studied at this stage—and cardiac effects. Because the pathophysiology of vascular and myocardial dysfunction during sepsis shares multiple pathways, we tested the hypothesis that (a) corticosteroids and/or APC improve sepsis-induced myocardial dysfunction in a cecal ligation and puncture (CLP) model of septic shock and that (b) this improvement involves downregulation of the iNOS pathway with a decreased production of radical oxygen species (ROS).
MATERIALS AND METHODS
Male Wistar rats weighing 380 to 420 g were obtained from the Centre d’élevage Depré (St Doulchard, France). All experiments were conducted in accordance with the National and European Institutes of Health guidelines for the use of laboratory animals and were approved by the University of Lorraine. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.
Rats were anesthetized with isoflurane (induction FI = 3% then 1.5%) with FIO2 = 60%. A 2-cm midline incision was made through the abdominal wall; the cecum was ligated at half the distance between the distal pole and the base of the cecum and subsequently punctured once with a 21-gauge needle. The cecum was slightly squeezed to expel a small amount of stool from the puncture site to ensure a full-thickness perforation. The bowel was returned to the abdominal cavity, and the incision was closed. A midline incision was also made in the anterior neck area. The right external jugular vein was catheterized (tubing PE-50) under sterile conditions and tunneled subcutaneously to the back of the neck. The exteriorized tubing was routed out of the animal cage through a helical stainless-steel wire spring to protect the tubing from manipulation by the animal and attached to a swivel device. The sham operation consisted of laparotomy and manipulation of the cecum without ligation or puncture. Neck surgery with catheterization for infusion was similar in the other groups except the control group, which did not undergo surgery (only conductance catheter studies were performed without any prior intervention).
During the first 4 h, rats were placed under a heating lamp until full awakening, and an analgesic was administered through the central line (nalbuphin 1 mg/kg over 2 min). Four hours after the surgery, all rats (except controls that were not instrumented) received antibiotics (imipenem 10 mg/kg over 10 min) and a continuous infusion of saline (10 mL/kg per hour) for a total duration of 14 h (from the fourth hour to the 18th hour). At this point, instrumented rats were randomly divided into six groups composed of five rats each: control, sham, peritonitis (CLP), dexamethasone (DEXA) 150 µg/kg per hour, recombinant human APC 33 µg/kg per hour (Lilly, Indianapolis, Ind), and DEXA + APC 150 µg/kg per hour–33 µg/kg per hour, diluted in the saline solution used for continuous fluid loading.
Surgical procedures for LV catheterization
Eighteen hours after surgery, rats were anesthetized again with isoflurane. The neck incision was reopened to expose the trachea and the left carotid artery. After intubating the trachea, the animal was then paralyzed (pancuronium 2 mg/kg) and placed on positive-pressure ventilation using a rodent ventilator (model 683; Harvard Apparatus SARL, Les Ulis, France) with FIO2 = 60%, respiratory rate = 53.5 * weight−0,26, tidal volume = 6.2 * weight1,01. A temperature probe was inserted into the rectum, and central body temperature was maintained between 36.7°C and 37.3°C using a heating pad and lamp. During the experiments, rats were perfused continuously with isotonic saline 10 mL/kg per hour. A bisubcostal incision was made to access the inferior wall of the diaphragm with an electrocautery system (MD 62; KLS Martin France SARL, Colmar, France). Thereafter, the diaphragm was opened, and a 3-0 suture was passed around the inferior vena cava (IVC) just above the diaphragm to perform IVC occlusions by pulling the suture. A 2F conductance catheter (SPR-838; Millar Instruments Inc, Houston, TX) was calibrated beforehand to convert raw arbitrary relative volume unit to true volume using calibration wells with known diameters (calibration cuvette 910–1048; Millar Instruments) containing heparinized blood from the same species of rat and heated at 37°C. Conductance catheter was inserted into the right carotid artery and advanced into the left ventricle. The proper positioning of the catheter was assessed by visual aspect of pressure-volume (PV) loops. The catheter was interfaced with a PV analog signal amplifier, and data were collected with an analog-to-digital converter (Millar Pressure Conductance Unit model 200; Millar Instruments Inc, Houston, Tex). All variables were displayed and recorded using IOX 18.104.22.168 software (EMKA Technologies SA, Paris, France). After a 10-min rest period, saline calibration was performed (40 µL hypertonic saline, 30%, bolus injection through central venous line) to estimate the parallel volume. After a further 10-min rest period, the LV pressure and volume were measured at steady state. A series of other parameters were also measured including heart rate, stroke volume, maximum pressure, CO, pressure development during isovolumic contraction (dp/dtmax), and relaxation (dp/dtmin). Upon completion of this step, IVC occlusion was performed by briefly lifting the ligature around the IVC to temporarily modify preload conditions. The LV end-systolic PV relationship (Ees) and end-diastolic PV relationship were obtained, as well as time-varying maximum elastance, preload-recruitable stroke work (PRSW), PV area (PVA), potential energy, and stroke work (SW). Just before the animals were killed, arterial blood was collected by heart puncture, allowing blood gas analysis. Plasma was isolated by centrifuging the blood immediately after collection and stored at −80°C. Finally, the myocardial ventricular tissue was sectioned for NO and superoxide measurements.
Electronic paramagnetic resonance studies
The electronic paramagnetic resonance (EPR) protocol has previously been described by Mostefai et al. (27) and Khoo et al. (28). Briefly, for superoxide anion spin trapping, right ventricles were treated for 45 min at 37°C with desferoxamine-chelated Krebs-HEPES solution containing 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH, 500 mmol/L; Noxygen, Elzach, Germany), desferoxamine (25 mmol/L), and diethyldithiocarbamate (DETC, 5 mmol/L). The reaction was stopped by putting the samples in ice. Samples were then frozen in liquid N2 and analyzed by EPR. For NO spin trapping, the technique was based on the use of colloid iron (II) DETC [Fe(DETC)2], to trap NO from left ventricles.
After testing for their normal distribution (Kolmogorov-Smirnov test), data are presented as mean (SD), where n represents the number of rats. Data were analyzed with statistical software GraphPad Prism version 4.0 (San Diego, Calif). Between-group differences were tested by one-way analysis of variance with Bonferroni correction. P < 0.05 was considered significant.
When compared with sham animals, CLP was associated with arterial hypotension (mean arterial pressure [MAP], 109 [SD, 12] vs. 77 [SD, 13] mmHg), no changes in heart rate, and increased lactate level (2.7 [SD, 0.8] vs. 5.7 [SD, 2.5] mmol/L) (Table 1).
CLP decreases systolic and diastolic myocardial function
Changes detected by conductance catheter readings confirmed that LV systolic function was impaired in CLP rats compared with sham animals (Table 2). Cardiac output was higher in septic animals, whereas LV maximal rate of pressure development (dP/dtmax) was significantly lower than that in sham-treated animals (7,213 [SD, 1,787] vs. 11,570 [SD, 1,242] mmHg/min, P < 0.001). Preload recruitable SW, a load-independent measure of systolic function, was significantly lower in peritonitis than in sham-treated animals (48 [SD, 18] vs. 104 [SD, 55] mmHg, P < 0.01). Similar results were found using the slope of the Ees. Conversely, ejection fraction was not different between these groups.
Furthermore, PV catheter readings obtained with this model accurately detailed abnormal changes in LV diastolic function in peritonitis. DP/dtmin was decreased by 38% and 37% during CLP in comparison with control and sham rats, respectively (P < 0.01). End-diastolic PV relationship was not statistically different between experimental groups.
CLP decreases myocardial efficiency
Pressure-volume area, an index of myocardial oxygen consumption, was significantly higher in CLP animals (42.1 [SD, 9.5] vs. 15.6 [SD, 6.6] mL/mmHg, P < 0.01). Nevertheless, mechanical efficiency (SW/PVA ratio) was significantly depressed during CLP (0.47 [SD, 0.07] in CLP rats compared with 0.67 [SD, 0.07] in control and 0.76 [SD, 0.12] in sham rats, P < 0.01).
Ventricular dysfunction is associated with increased NO and superoxide anion production
Myocardial NO and superoxide anion levels were significantly higher in the CLP group than in controls or sham-treated rats (Fig. 1).
Comparative effects of APC, DEXA, and their combination
DEXA, APC, and their combination thereof improve systemic hemodynamics
Mean arterial pressure was restored by DEXA and APC + DEXA. A nonsignificant improvement in MAP (P = 0.09) was observed in APC-treated rats when compared with CLP rats. Cardiac output was restored to basal values with all treatments. Lactate levels decreased in all treated groups (Table 1).
DEXA, APC, and their combination thereof improve ventricular function and cardiac efficiency
Sepsis-induced decreases in dP/dtmax, dP/dtmin (first derivative of minimal developed pressure during isovolumetric relaxation), Ees, and PRSW were significantly corrected by all treatments (Table 2). Conversely, values of ejection fraction were similar between all groups. Treatments decreased myocardial oxygen consumption and improved myocardial mechanical efficiency (Table 3).
Improvements in myocardial function are associated with a decrease in myocardial NO and superoxide anion
Lastly, myocardial NO and superoxide anion levels decreased in all treated groups except for superoxide anion in the DEXA group. Nonetheless, addition of DEXA to APC enabled an even greater decrease in NO and superoxide anion levels than with APC alone, reaching levels observed in sham and control animals (Fig. 1).
Characterization of the links between inflammation, coagulation, and the immune and neuroendocrine systems along with randomized studies has led to international recommendations for the use of low-dose hydrocortisone and, until recently, the use of recombinant human APC in the early management of septic shock (15). In the present study, we report that APC and DEXA improve sepsis-induced cardiac dysfunction. These effects were associated with an important decrease in NO and ROS production.
Because the endotoxemia model is probably far from the clinical setting, we used a more relevant protocol of CLP, combining volume resuscitation, antibiotics, and delayed treatment (4 h after CLP induction). The use of a high-volume fluid therapy led to a hyperdynamic model and represents the large majority of resuscitated septic patients. Moreover, close to human septic shock and despite a hyperkinetic state, CLP rats displayed a systolodiastolic myocardial dysfunction associated with an increase in myocardial oxygen consumption leading to a decrease in myocardial efficiency. Importantly, as extensively described (29), concerns about the ability of ejection fraction to identify cardiac dysfunction were clearly outlined. Altered contractility combined with low afterload in the CLP group has certainly masked changes in ejection fraction. Finally, we observed an association between decreased myocardial function and myocardial NO and superoxide anion overproduction.
APC and DEXA improve myocardial function
We showed that both treatments improved systolic and diastolic dysfunction. Regarding APC, this is consistent with the findings of Favory et al. (30), who reported a correction of ex vivo systolic dysfunction with murine APC infusion during experimental endotoxinic shock. In this study, intrinsic contractile cardiac function was assessed by means of isolated heart perfusion, a method that informs about contractile function but that cannot take into account the peripheral influences from nervous or humoral systems, unlike conductance catheter studies. In this latter study, beneficial effects of APC were also associated with attenuation of entotoxin-induced inflammatory response and a reduction of plasma levels of nitrite/nitrate.
Similarly, it is now well recognized that corticosteroids during septic shock improve shock outcome in the most severe patients (31). Dexamethasone has been shown to improve arterial contractility and endothelial dysfunction in septic shock rats (26). Nevertheless, data concerning myocardial effects of glucocorticoids during septic shock are scarce. Using hydrocortisone, Feng et al. (32) noted an improvement in dP/dtmax in CLP-induced septic rabbits, whereas Wang et al. (33) observed a decrease in myocardial NO and iNOS expression, as well as troponin levels in septic rats. In the present study, using relevant parameters of systolic and diastolic function, we found that DEXA improved systolic and diastolic function. Similarly to human septic shock, we observed a marked decrease in sepsis-induced hyperkinetic state with both drugs (34). This effect may be related to an increase in afterload secondary to the improvement in vascular reactivity but also to the decrease in myocardial oxygen consumption.
APC and DEXA improve cardiac efficiency
Mechanical efficiency has long been assessed by the SW/PVA ratio (35). The SW/PVA ratio reflects the mechanical efficiency of converting the total mechanical energy (PVA) available to the left ventricle to external work (SW). In our study, we found a significant alteration of SW/PVA ratio during CLP, which was normalized after APC and/or DEXA infusion. Septic shock induced a severe alteration of energetic myocardial efficiency, related to an important increase in energy consumption in excitation-contraction coupling, as previously reported (36). During severe sepsis, the heart is in a state of considerable “oxygen waste” due to increased energy utilization for electronic work, most likely partly secondary to decreased efficiency of the mitochondria (36). Peroxynitrite has been involved in the alteration of almost every component of the mitochondrial electron transport chain through mechanisms involving cysteine oxidation, tyrosine nitration, and damage of iron sulfur centers (37). Although we only indirectly estimated peroxynitrite formation in the myocardium, we can hypothesize that improvement in cardiac efficiency observed after APC and/or DEXA infusion is at least partly mediated by inhibition of ROS production and specifically peroxynitrite in the heart. This hypothesis was confirmed by Lancel et al. (12), who found an improvement in myocardial contractile dysfunction in endotoxin-treated rats with the use of peroxynitrite neutralizers.
Effects on ROS production
Although we did not extensively investigate the pathways implicated in sepsis-induced cardiac dysfunction, our results confirm that septic cardiomyopathy is associated with an overproduction of NO and superoxide anion and that both APC and DEXA decreased this production. In a previous study, we similarly demonstrated that these two drugs also decreased NO and superoxide anion overproduction at the vascular level. Peroxynitrite is known to play a major role in sepsis-induced cardiomyopathy. Although we did not evaluate peroxynitrite production at the cardiac level, it is well demonstrated that when both superoxide and NO are synthesized within a few cell diameters of each other, they combine spontaneously to form peroxynitrite by a diffusion-limited reaction (37). So we can readily hypothesize that limitation of peroxynitrite formation is at least partly responsible for our findings on improvement of sepsis-induced cardiac dysfunction by APC and DEXA.
When considering hemodynamic or contractility parameters, administration of APC or DEXA alone allowed almost a complete restoration of these parameters to the level of control ones. Identifying a further effect in the APC + DEXA group was then unlikely. Nevertheless, NO and superoxide anion levels in the heart were decreased by APC or DEXA alone, and coadministration induced a much more reduction, suggesting an additive effect.
First, on October 25, 2011, Eli Lilly and Company announced the voluntary withdrawal of Xigris (drotrecogin alfa [activated]) following the negative results of its most recent clinical trial, the PROWESS-SHOCK study (19). Despite these disappointing results, there were numerous preclinical studies demonstrating potential beneficial effects of APC. Thus, even if the present results do not lead to further progress in the clinical area, they are nonetheless valuable from a pathophysiologic point of view. Moreover, many reasons other than product inefficiency may explain a negative result. An unfavorable benefit-risk balance related to hemorrhagic complications may have mitigated the undeniable effects of APC, and the development of new molecules sharing the same beneficial pathways without anticoagulant drawback should be encouraged.
Second, and because APC and corticosteroids display a myriad of genomic and nongenomic effects, we were not able to explore all implicated cellular pathways. We therefore chose to focus on ROS production and in particular on NO and superoxide anion. Many publications have identified APC and corticosteroids as iNOS inhibitor, and there is mounting evidence against the deleterious role of iNOS in sepsis-induced organ dysfunction. However, ischemia/reperfusion phenomenon is a contributor of organ dysfunction during septic shock, and the role of iNOS during ischemia/reperfusion is much more controversial (38). Moreover, a recent study has pointed out the participation of mitochondrial NOS in sepsis-induced cardiomyopathy (39). As our work was not designed for it, we were not able to differentiate between these several isoforms of NOS.
Finally, the choice of DEXA, which is fivefold more anti-inflammatory than hydrocortisone, as well as the dose delivered should be discussed. In clinical practice, low-dose corticosteroid therapy is preferred to high doses because high dosages have been associated with an increase in the risk of death (31). Nevertheless, when reviewing the experimental literature, all of the studies reported an improvement in survival independently of the molecule, the dose, and the presence of mineralocorticoid effect. This may be explained by the fact that the deleterious effects of high doses in clinical setting are generally related to infections that are not present in a short and lethal model of septic shock.
Activated protein C and corticosteroids improve not only sepsis-induced vascular dysfunction but also sepsis-induced cardiac dysfunction and myocardial energetic efficiency in a rat model of hyperdynamic septic shock. These effects are mediated by the limitation of NO and superoxide anion production in the myocardium.
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