Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Septic shock, a major cause of high mortality rate in intensive care units, is usually associated with multiple organ failure, including hypercapnic respiratory failure (1). Bacterial lipopolysaccharide (LPS), the outer membrane of gram-negative bacteria, is widely accepted as a central player in the pathogenesis of septic shock by activating the release of mediators and cytokines from various cells. Several groups of investigators have confirmed that in vitro and in vivo exposure to LPS elicits a significant decline in the contractile function of the ventilatory and limb muscles (2, 3). The exact mechanism of depressed muscle contractility in septic shock remains under investigation; however, several local mediators have been implicated, such as prostaglandins, thromboxanes, reactive oxygen species, cytokines, nitric oxide, and, lately, endothelins (ETs) (3, 4).

ETs are a family of acidic 21-amino-acid peptides found in at least three distinct isoformsET-1, ET-2, and ET-3 which share sequence homology and arise through proteolytic processing of precursor prohormones (preproETs) (5). PreproETs are proteolytically cleaved to form proETs (Big ETs) which are between 38 and 41 amino acid peptides (5). These, in turn, are cleaved by two isoforms of enzymes known as endothelin-converting enzymes (ECE-1 and ECE-2) to form mature ET peptides. ETs exert numerous biologic actions by acting through two main G-protein coupled receptor families, ET-A and ET-B receptors. Both of these receptors stimulate phospholipase C, which leads to increased formation of diaceylglycerol and inositol-1,4,5-trisphosphate, which, in turn, activates protein kinase C pathway and increases intracellular Ca⁺⁺, respectively (6).

It has been well established that circulating ET-1 levels increase significantly in septic humans and in LPS-injected animals (7). Both increased local production of ETs and poor pulmonary and renal clearance of ETs have been blamed for the rise in circulating ETs in septic shock. The exact role played by ETs in the pathogenesis of septic shock remains under investigation. Many investigators, however, have proposed that ETs have deleterious effects on hemodynamics and organ function in septic shock because of maldistribution of blood flow as a result both of severe vasoconstriction in certain vascular beds and of enhanced release of vasodilators such as NO and PGI₂ from other organs. Maldistribution of blood flow is expected to influence directly the normal function of various organs, including kidneys, liver, and heart (12). In addition, there exist several pathways through which ETs could negatively influence skeletal muscle contractile performance. These include direct activation of muscle ET-A receptors (13), enhanced release of prostaglandins and reactive oxygen species (14, 15), and finally, reduction in blood flow as a result of severe constriction of arterioles and venules (16). Despite the possible involvement of ETs in the regulation of skeletal muscle function, regulation of local ET production in these muscles under normal and pathologic conditions, such as septic shock, has largely been ignored.

In this study, we investigated whether local production of ET-1 in the ventilatory muscles is altered in response to septic shock. We also assessed whether ET production in the ventilatory muscles is regulated through changes in the gene expression of ET precursors and/or expression of ECE enzymes. For the first time, our results indicate that LPS injection is associated with a significant and prolonged rise in diaphragmatic ET concentration and that this response is due to upregulation of preproETs as well as enhanced ECE-1 expression. We also found that endothelial cells as well as somatic muscle fibers were the sources of enhanced ET production in septic animals.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Animal Preparation

The procedures for the care and use of animals were approved by the Animal Care Committee of the Royal Victoria Hospital (Montreal, PQ, Canada). Four groups (n = 6 in each group) of pathogen-free male Sprague-Dawley rats (250-300 g) were housed in the animal facility of the Royal Victoria Hospital and were studied 1 wk after arrival. Group 1 was injected with normal saline (control group). Groups 2, 3, and 4 were injected i.p. with Escherichia coli LPS (serotype 055:B5, 20 mg/kg; Sigma, Inc., St. Louis, MO) and killed by cervical dislocation 6, 12, and 24 h after the injection, respectively. The diaphragm was dissected and quickly frozen in liquid nitrogen. For immunostaining, the diaphragm was sandwiched between liver slices and flash-frozen in cold isopentane (20 s), then immersed in liquid nitrogen and stored at 80°C.

Tissue ET-1 Measurement

Details of the technique have been published previously (17). In brief, 100- to 200-mg diaphragmatic samples were homogenized in 4 M guanidine thiocyanate/0.1% trifluoroacetic acid on ice (3 bursts of 10 s). An aliquot was taken for protein measurement (Bio-Rad, Inc., Hercules, CA). The homogenate was then centrifuged at 5,000 × g for 20 min at 4°C. The supernatant was then loaded on a Sep Pak C18 cartridge. The cartridge was washed with 4 ml of H₂O and 4 ml of 20% methanol. The absorbed peptides were then eluted with 90% ethanol. Recovery of the whole procedure was expected to be 66%. Tissue ET-1 was then detected with radioimmunoassay established in our laboratory (18). Diaphragmatic ET-1 content was expressed as picograms per gram of wet tissue weight.

Immunohistochemistry

Air-dried cryostat sections (10 µm) were rehydrated with phosphate-buffered saline (PBS) (pH 7.4) for 3 to 5 min and then blocked for 1 h with normal goat serum, followed by washing with PBS. For accurate detection of ET-1, we used polyclonal antibody raised against the C-terminal of ET-1 (19). This antibody was previously used to detect ET-1 in a variety of species (19). Sections were incubated with primary antibody for 2 h at room temperature. After three washes in PBS, sections were incubated with biotinylated goat antirabbit secondary antibody followed by the avidin-biotin-peroxidase complex (Vector Laboratories, Burlingame, CA). Sites of immunoreaction were visualized by immersing sections in a solution of diaminobenzidine and hydrogen peroxide. Counterstaining was performed with hematoxylin (Sigma). Negative controls were prepared with primary antibodies absorbed with the cross-reactive endothelins.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

This technique was used to assess changes in messenger RNA (mRNA) levels of preproET-1, preproET-3, and ECE-1. Total RNA was extracted from diaphragmatic samples following the method described by Chomczynski and Sacchi (20). Total RNA was reverse-transcribed using random hexamers and murine Moloney Leukemia virus reverse transcriptase (Life Technologies, Gaithersburg, MD). RT-generated complementary DNA encoding preproET-1, preproET-3, ECE-1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (both as an internal and positive control) were amplified with PCR. RNA with no clear GAPDH band in the RT-PCR products (30 cycles) was discarded from further studies. Oligonucleotide primers (synthesized in McGill University DNA synthesis facility, Montreal, PQ, Canada) for preproET-1, preproET-3, and ECE-1 were based on the sequence used by Wang and colleagues (21) (Table 1). Experimental conditions for all PCR reactions were: initial denaturation at 95°C for 5 min followed by 35 cycles (94°C 1-min denaturation, 54°C 1-min annealing, and 72°C 1.5-min extension). This was followed by a final 72°C 10-min extension. Ethidium bromide-stained 2% agarose gels were used to separate PCR products. PCR products were visualized under ultraviolet light and the optical densities of DNA bands were scanned with a BioRad image densitometer and quantified using SigmaGel software (Jandel Scientific, San Rafael, CA). To verify the accuracy of the amplified sequence, PCR products were cloned in PCRII plasmid (using a TA cloning kit from Invitrogen, San Diego, CA) and sequenced in the McGill University DNA sequencing facility. In order to use RT-PCR semiquantitatively, we assessed the relationship between total RNA concentration per sample and the optical density of PCR product by varying RNA concentrations and fixing PCR cycle number at 35 cycles. We chose to study total RNA concentrations of 100 ng for preproET-1 and preproET-3, and 250 ng for ECE-1 amplification, on the basis of the relationship between total RNA concentration obtained from normal rat diaphragm and the optical density of ethidium bromide-stained PCR products after 35 cycles (Figure 1). For GAPDH , we used 50 ng of total RNA.

View larger version (13K): [in this window] [in a new window]   Figure 1.   The relationship between total diaphragmatic RNA concentration (x axis) and optical density of PCR products after 35 cycles of PCR using oligonucleotide primers selective to preproET-1 (left) and preproET-3 (right). Note the range of the linear portion of the amplification process.

Data Analysis

Results of ET-1 concentration are presented as mean ± standard error of the mean (SEM). Differences in ET-1 concentration between control and LPS groups were compared using two-analysis of variance for repeated measures. Any differences detected were evaluated post hoc using the Neuman-Keuls procedure. P < 0.05 was considered significant. Similar analysis was used to compare optical density of RT-PCR products stained with ethidium bromide.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Figure 2 illustrates the average values of diaphragmatic ET-1 concentration in control and septic animals. Injection of LPS elicited a prolonged and a significant rise in diaphragmatic ET-1 concentration, which remained higher than control values 24 h after LPS injection (Figure 2).

View larger version (33K): [in this window] [in a new window]   Figure 2.   Diaphragmatic tissue ET-1 concentration in the control animals and animals killed after 6, 12, and 24 h of E. coli LPS injection. **P < 0.01 compared with control values.

Figure 3 shows the size of RT-PCR products obtained from total diaphragmatic RNA by using oligonucleotide primers selective to rat preproET-1, preproET-3, and ECE-1 mRNA transcripts. Cloning of these products confirmed the identity of these transcripts. LPS injection elicited a significant and early (within 6 h) rise in mRNA expression of diaphragmatic preproET-1, preproET-3, and ECE-1 (Figure 4). After 24 h of LPS injection, the expression of these genes remained higher than control values. Figure 5 illustrates the mean values (± SEM) of optical density of three independent RT-PCR measurements on three separate samples of diaphragmatic RNA. Whereas mRNA levels of preproET-1 and preproET-3 rose by about 4- and 3-fold within 6 to 12 h of LPS injection (P < 0.01 compared with control), mRNA of ECE-1 increased by more than 10-fold and peaked within 24 h of LPS injection (P < 0.01 compared with control values). GAPDH mRNA after endotoxin injection remained similar to control values (Figure 5).

View larger version (72K): [in this window] [in a new window]   Figure 3.   The size of RT-PCR products obtained from total diaphragmatic RNA by using oligonucleotide primers selective to rat preproET-1, preproET-3, and ECE-1 mRNA transcripts. Cloning of these products confirmed the identity of these transcripts.

View larger version (40K): [in this window] [in a new window]   Figure 4.   The influence of septic shock on mRNA expression of diaphragmatic preproET-1 (top), preproET-3, ECE-1, and GAPDH (bottom) in the control and LPS-injected rats. Note the significant induction of preproET-1 and preproET-3 in LPS-injected animals. Also notice that only weak ECE-1 mRNA expression was detected in the control animals, whereas significant expression was detected after LPS injection.

View larger version (32K): [in this window] [in a new window]   Figure 5.   Changes in the mean values of optical densities of three independent RT-PCR measurements on three separate samples of diaphragmatic RNA samples. Notice that whereas upregulation of preproET-1 and preproET-3 mRNA was detected early (within 6 and 12 h after LPS injection), the rise in ECE-1 mRNA expression was progressive and peaked 24 h after LPS injection.

Figure 6 shows immunostaining of rat diaphragm with anti-ET-1 antibody. Positive staining (arrows) was detected in the endothelium (upper right panel ) and muscle fibers (upper and lower left panels) of diaphragm obtained from LPS-injected rats. None of the muscle fibers of control rats stained positively for ET-1 (lower right panel ). Preincubation of primary antibody with authentic ET-1 completely eliminated the positive staining observed in the LPS-injected rats (results not shown).

View larger version (114K): [in this window] [in a new window]   Figure 6.   Immunostaining of the rat diaphragm with anti-ET-1 antibody. Positive staining (brown color, arrows) was detected in the endothelium and muscle fibers in the diaphragm after 12 h of LPS injection (upper right and left, and lower left). None of the muscle fibers of the control rats (lower right) stained positively for ET-1. The selectivity of the primary antibody was confirmed by preincubation with authentic ET-1 (results not shown).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The main finding of this study is that endotoxin injection elicits a significant rise in ET-1 production in the diaphragm as a result of upregulation of both ET-1 precursor (preproET-1) and ECE-1 expressions. Endothelial cells as well as striated muscle fibers were the main sites of enhanced diaphragmatic ET-1 production in septic animals. We also found that endotoxin injection leads to enhanced diaphragmatic ET-3 production, as indicated by the significant rise in preproET-3 mRNA expression.

Production of ETs in Septic Shock

It has been well established that both cytokines and LPS elicit a significant rise in ET-1 production by the cultured endothelial cells (22). Subsequent reports confirmed that other cells, such as macrophages, cardiac myocytes, airway epithelial cells, and mesangial cells, are capable of releasing ET-1 in response to in vitro cytokines and LPS exposure (23). In animals with septic shock, several authors described a significant and early rise in circulating ET-1 and Big ET-1 levels (7). In patients with septic shock, plasma ET-1 concentration correlated positively with the severity of the illness (9, 11) and the rise in pulmonary arterial pressure (9), and negatively with systemic arterial pressure (11). Increased ET-1 production by various cells, as well as impaired renal and pulmonary clearance of ET-1, have been proposed as the mechanisms responsible for the rise in circulating ET-1 in septic shock.

Although the exact roles played by different ETs in the peripheral vascular failure of septic shock remain speculative, several authors have asserted that relatively high levels of ET may play a deleterious role in causing the maldistribution of blood flow and multiple organ failure observed in septic patients. This notion is supported by the observations that infusion of anti-endothelin antibody in septic rats improves kidney function (27) and increases mesenteric blood flow (28), and by the observation that pretreatment with ET receptor antagonists reduces LPS-induced pulmonary hypertension in pigs (7).

Production of ETs by Skeletal Muscles

Unlike other cell types, the possibility that skeletal muscle fibers could also synthesize and release ETs has not been investigated in detail. Instead, investigators recognized skeletal muscle fibers as a target for ET action because muscle fibers possess abundant ET receptors (29). Activation of these receptors by ET-1 enhances glucose uptake and mobilizes Ca⁺⁺ in cultured human and rat myocytes (30). It has also been proposed that skeletal muscles may also be involved in the degradation of ET-1 because of the presence of the neutral endopeptidase-24.11 (31). To the best of our knowledge, we are the first to show ET-1 synthesis by skeletal muscle fibers in septic shock. Our RT-PCR results suggest that ET-3 synthesis is also enhanced in muscle fibers during the course of septic shock. These results contrast with those of Kaddoura and associates (32), who reported no change in skeletal muscle prepro-ET-1 mRNA expression after 6 h of LPS injection in rats. The reasons for the differences between our study and that of Kaddoura and coworkers (32) are unclear but we speculate that differences in the type of muscle and rat strains may be involved. The type of skeletal muscle studied by these investigators was not identified. Moreover, their failure to detect changes in preproET-1 mRNA expression may be due to the lower sensitivity of ribonuclease protection assay used by these investigators as compared with the RT-PCR used in our study. In addition to the use of a sensitive technique to detect changes in mRNA expression, we confirmed that upregulation of preproET-1 mRNA was associated with increased ET-1 protein by measuring tissue concentration and localization of ET-1 in the diaphragm, whereas only mRNA measurement was performed by Kaddoura and colleagues (32).

ECE-1 is broadly distributed in a variety of tissues, as evidenced by abundant mRNA expression in the lungs, pancreas, placenta, ovary, testis, and brain (33, 34). In the majority of these tissues ECE-1 is localized to the endothelial cells, although it has also been detected in secretory cells, such as adrenal cells (34). Our results indicate that ECE-1 mRNA is expressed at relatively low levels in the normal diaphragm. However, LPS injection elicits a substantial rise in ECE-1 mRNA expression which far exceeded the rise in preproET-1 and preproET-3 expressions. Thus, in vivo endotoxemia appears to provoke a coordinated upregulation of both ET precursors and the key biosynthetic enzyme responsible for the conversion of Big ET-1 to ET-1. In addition, for the first time, our results indicate that ECE-1 expression is regulated in response to LPS injection. Other investigators reported that ECE-1 mRNA expression remains unchanged in response to inflammatory mediators (35).

In the current study we did not address the mechanisms leading to upregulation of diaphragmatic ET production in septic animals; however, we speculate that several mediators (such as LPS, pro-inflammatory cytokines such as tumor necrosis factor- and interleukin-1, thrombin, coagulation products, products of activated leukocytes, platelet activating factor, hypoxia, and changes in shear stress) may be operating during the course of septic shock, leading to enhanced release of ETs from the endothelial cells and skeletal muscle fibers.

Implications

Our finding that diaphragmatic ET-1 production is significantly elevated in septic animals implies that the diaphragm could be an important source of the elevated circulating ET levels observed in septic shock. Another implication of our results is that enhanced ET-1 released by muscle fibers and endothelial cells could act in an autocrine fashion to compromise diaphragmatic contractile function. This effect is likely to be mediated through the proinflammatory role of ET-1, which includes promotion of neutrophil aggregation; activation of mast cells, macrophages, and monocytes leading to enhanced prostaglandin and reactive oxygen species release; and finally, stimulation of adhesion molecule expression on the endothelial cells (14, 15, 36, 37). ET-1 could also depress diaphragmatic contractile performance by acting directly on ET-A receptors located on muscle fibers, leading to depression of diaphragmatic contractility (13). Another likely pathway through which ETs may negatively influence diaphragmatic contractility is reduction of blood flow. Activation of ET-A receptors by ET-1 elicits strong and prolonged constriction of large-bore arteries, arterioles, and venules of in situ isolated limb muscles (16). It is possible, therefore, that endogenous ET release plays an important role in the maldistribution of blood flow and depressed diaphragmatic contractility frequently observed in animal models of septic shock. More research is needed to document the contribution of ETs to sepsis-induced ventilatory muscle failure.

In summary, our results indicate, for the first time, that injection of endotoxin is associated with increased production of ETs by diaphragmatic muscle fibers and endothelial cells lining diaphragmatic vasculature. Enhanced ET production is accomplished through upregulation of both ET precursors as well as endothelin-converting enzyme.