INTRODUCTION
In classic ischemic preconditioning (IPC), brief periods of coronary artery occlusion-reperfusion, transiently (10–120 min) protects the myocardium against subsequent lethal ischemia-reperfusion injury (1). After dissipation of this acute protection, a second window of protection (2) or delayed preconditioning (DP) appears 12–24 h later, which lasts up to 72 h. In addition to reducing myocardial infarct size (2–5), DP enhances the resistance of the myocardium against myocardial stunning (6), arrhythmias (7), and post-ischemic endothelial dysfunction (8).
Several triggers induce pharmacological DP in the heart including wall fragments of gram-positive bacteria such as lipoteichoic acid (LTA) (5), wall fragments of gram-negative bacteria such as lipopolysaccharide (LPS or endotoxin) (4,9), the structurally related monophosphoryl lipid A (MLA) (10), or the novel glycolipid RC-552 (11,12). LPS, MLA, and RC-552 contain the lipid A moiety, which mediates many of the biological effects of LPS. Although RC-552 is less toxic than MLA (and MLA is many times less toxic than LPS), recent data from knockout mice has provided evidence that RC-552 is still an immunomodulator, which causes upregulation of the inducible nitric oxide synthase (iNOS) (12).
Gram-positive organisms, however, do not contain lipid A or LPS, which is the cell wall component of gram-negative bacteria responsible for the initiation of septic shock. Gram-positive bacteria such as S. epidermidis can also cause septic shock and multiple organ failure without causing endotoxemia (13), and endotoxin is not always found in the serum of patients with septic shock (14). The cell wall of gram-positive bacteria contains LTA and peptidoglycan (PepG), a large polymer, which provides stress resistance and shape-determining properties to bacterial cell walls. LTA is a macroamphiphile molecule, which is equivalent to LPS in gram-negative bacteria (15). The predominant type of LTA contains a 1,3-linked poly-(glycerophosphate) chain attached by a phosphodiester bond to a glycolipid or phosphatidyl-glycolipid, which is a characteristic membrane lipid of gram-positive bacteria (15). The lipid moiety on the LTA renders LTA amphiphilic and enables it to anchor to the cytoplasmic membrane by hydrophobic interaction. The glycolipids differ in structure among gram-positive bacteria, usually in a genus-specific manner, and the glycolipid moieties of LTA that are derived from them vary accordingly (15). S. aureus is a major cause of nosocomial infections and gram-positive septic shock (14), whereas B. subtilis is a nonpathogenic gram-positive bacterium (16,17). The molecular structures of LTA from S. aureus and B. subtilis are very similar, except that the glycosylation and alanine substitution of the poly-glycerophosphate backbone is higher in S. aureus (15). This substitution changes the chemical features of LTA. The wall fragment is more lipophilic and much more toxic in several different disease models compared with the LTA derived from B. subtilis.
Since the discovery in 1983 of the mitochondrial KATP (mitoKATP) channel in isolated pig ventricular myocytes (18), numerous studies have investigated the effects of drugs that open or close this channel in conditions associated with myocardial ischemia (for review see Refs. 19 and 20). Today, we know that mitoKATP channels are involved in the beneficial effects of IPC. Inhibitors of the mitoKATP channel, such as glibenclamide (inhibitor of sarcolemmal and mitochondrial KATP channels) or 5-hydroxydecanoate acid (5-HD, inhibitor of mitoKATP channels), abolish the cardioprotective effects of IPC, whereas openers of mitoKATP channels, such as diazoxide, aprikalim, or bimakalim, mimick the beneficial effects of IPC (for review see Ref. 20). Activation of transmembrane receptors, such as adenosine (21) or opioid (22), causes the opening of mitoKATP channels and cardioprotection. The mechanism by which opening of mitoKATP channels protects the myocardium against ischemic injury is not entirely clear.
Because it was shown that MLA produces a similar reduction in infarct size as IPC, it was hypothesized that the mitoKATP channel might be an important effector of IPC and/or DP. Therefore, by using glibenclamide and/or 5-HD it was shown that the cardioprotective effects of MLA were abolished in the late phase of preconditioning (23). In fact, MLA also induces iNOS with enhanced release of NO, which directly activates mitoKATP channels and may cause cardioprotection (24,25). There is also evidence that mitoKATP channels are involved in the delayed cardioprotective effect of adenosine A1 receptor activation (21). Most investigators used the specific mitoKATP channel inhibitor 5-HD (at a dose of 5 mg/kg i.v.) to study the role of mitoKATP channels in IPC and/or DP (21,26).
Currently, not much is known about DP induced by bacterial wall fragments in the heart (5). Therefore, we investigated the delayed cardioprotective effects of LTA derived from the nonpathogenic gram-positive bacterium B. subtilis in the rat in vivo. Furthermore, we studied whether DP afforded by LTA from nonpathogenic and pathogenic strains of gram-positive bacteria is mediated by late opening of mitoKATP channels.
MATERIALS AND METHODS
Male Wistar rats (200–330g) were obtained from Tuck (Rayleigh, Essex, UK) and cared for according to AAALAC guidelines and Guide for the Care and Use of Laboratory Animals (Department of Health and Human Services, National Institutes of Health, Publication No. 86-23). Animals were housed for at least 3 days in a stress-free environment after transportation from the supplier. All procedures were conducted in accordance with the Home Office Guidance on the Operation of Animals (Scientific Procedures) Act 1986 published by HMSO, London.
Myocardial ischemia-reperfusion in the rat
Myocardial injury was caused by occlusion-reperfusion (25–120 min) of the left anterior descending coronary artery (LAD). Rats were randomly allocated into the groups described in Table 1 and injected IP either 16 h before 1) no occlusion of the LAD (sham) or 2) LAD occlusion-reperfusion (I-R). The technique used to produce LAD occlusion was identical to that described previously (4,5). Rats were anesthetized by using sodium thiopentone (120 mg/kg i.p.). The trachea was cannulated and artificial respiration was maintained by using a Harvard ventilator. The right carotid artery was cannulated to monitor mean arterial blood pressure (MAP) and heart rate (HR). Pressure rate index (PRI), a relative indicator of myocardial oxygen consumption (27), was calculated as the product of MAP and HR and expressed in mmHg/(min · 103). The right jugular vein was cannulated for the administration of drugs. The chest was opened by a left-side thoracotomy, the pericardium was incised, and an atraumatic needle and occluder were placed around the LAD. After completion of the surgical procedure, the animals were allowed to stabilize for 30 min before LAD ligation. After 25 min of acute myocardial ischemia, the occluder was released, allowing the reperfusion of the previously ischemic myocardium for 2 h. After re-occluding the LAD, Evans blue dye (1 mL of 2% wt/vol) was administered i.v. to distinguish between ischemic (area at risk [AR]) and nonischemic myocardium (area not at risk). Subsequently, the heart was cut into horizontal slices and then into small pieces. The AR was separated from the area not at risk and then incubated with p- nitro-blue tetrazolium (0.5 mg/mL, 20 min at 37°C) to distinguish between ischemic and infarcted tissue (28), whereas the area not at risk was incubated with saline. The AR and infarct size were calculated after weighing the respective tissue samples and expressed as percent of the AR.
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Table 1: Animal groups exposed to bacterial products followed by occlusion-reperfusion (25–120 min) of the left anterior descending coronary artery (LAD) or sham surgery
Materials
Unless otherwise stated, all compounds were obtained from Sigma Chemical Co. (St. Louis, MO). LTA was obtained by a phenolic extraction procedure from S. aureus or B. subtilis (29). Intraval (sodium thiopentone) was obtained from May & Baker Ltd.
Statistical analysis
Data are reported as means ± SEM of n observations. ANOVA followed by a Dunnett's post test was used to compare data. A value of P < 0.05 was considered statistically significant.
RESULTS
Effects of bacterial wall fragments on myocardial infarct size
The mean values for the AR were similar in all animal groups studied and ranged from 44 to 53% (P > 0.05, data not shown). In rats pretreated with saline, LAD occlusion-reperfusion (25–120 min) resulted in an infarct size of approximately 60% of the AR. Compared with saline, administration of LTA from S. aureus or B. subtilis 16 h before coronary artery ligation caused significant reductions in myocardial infarct size (P < 0.05;Fig. 1). Compared with LTA (both strains of bacteria) treatment alone, 5-HD did not inhibit the infarct size reduction afforded by LTA. Sham operation with saline, 5-HD, LTA (S. aureus), or LTA (B. subtilis) alone did not result in a significant degree of infarction in any of the animal groups studied (<3% of the AR, P > 0.05, data not shown).
Fig. 1: Myocardial injury caused by occlusion-reperfusion (25 min-2 h) of the left anterior descending coronary artery (LAD). Rats received saline (1 mL/kg i.p.) or LTA from either S. aureus (1 mg/kg) or B. subtilis (1 mg/kg) 16 h before LAD occlusion-reperfusion (I-R). 5-hydroxydecanoate (5-HD, 5 mg/kg) was administered i.v. 10 min before I-R. Infarct size is expressed as percent of the area at risk (AR). Data are expressed as means ± SEM of n observations. ANOVA followed by a Dunnett's post test was used to compare data. * P < 0.05 compared with saline control.
Hemodynamic data from rats subjected to myocardial ischemia-reperfusion
Hemodynamic data, e.g., MAP, HR, and PRI measured during the course of the experiments were similar in all groups studied (P > 0.05;Table 2). In sham-operated rats (no LAD occlusion), injection of either saline, 5-HD, LTA (S. aureus), or LTA (B. subtilis) did not cause any significant effects on MAP, HR, or PRI (P > 0.05, data not shown). In rats subjected to LAD occlusion-reperfusion, which were subjected to either saline, 5-HD, LTA (S. aureus) or LTA (B. subtilis) treatment, mean values for MAP and PRI fell throughout the experiment (P < 0.05;Table 2).
Table 2: Mean arterial blood pressure (MAP), heart rate (HR), and pressure rate index (PRI) measured during myocardial ischemia-reperfusion
DISCUSSION
This study shows for the first time that pretreatment (16 h before LAD occlusion-reperfusion) with LTA from the nonpathogenic gram-positive bacterium B. subtilis protects the heart against a subsequent period of regional ischemia-reperfusion. We used LTA from the pathogenic gram-positive bacterium S. aureus (1 mg/kg) as a recognized control with which to induce DP (5). Recently, we showed that the effects of LTA from S. aureus are time dependent and accompanied by a reduction of cardiac troponin T release into the plasma and a reduction in the histological signs of necrosis in this model of myocardial ischemia-reperfusion (5). LTA from B. subtilis (1 mg/kg i.p.) reduced myocardial infarct size to the same degree as LTA from S. aureus (∼60% reduction in infarct size). B. subtilis is a less pathogenic gram-positive bacterium than S. aureus and, therefore, may offer a new therapeutic approach to exploit DP.
Clearly, in all groups studied, there were no significant differences in body weight, heart weight, AR, or hemodynamic parameters such as MAP or HR, suggesting that the beneficial effects of the different bacterial wall fragments were not related to differences in the amount of myocardial tissue sampled nor to changes in myocardial oxygen demand. What, then, are the mechanisms by which bacterial wall fragments induce DP in the rat heart in vivo? We and others recently reported that different pathways, e.g., NO (30–32), iNOS (33), antioxidative enzymes (5), or protein kinase C (34) are involved in DP. For instance, LPS causes a release of cytokines such as tumor necrosis factor (TNFα) or interleukin (IL-1β) and, hence, an upregulation of the Mn-SOD gene (5). In contrast, the same study showed that LTA from S. aurues was not sufficient to induce the Mn-SOD gene.
However, in this study we investigated whether the mitoKATP channel is an important end effector in DP afforded by LTA from S. aureus or B. subtilis. Opening of mitoKATP channels is protective in ischemic heart, and the mechanism of cardioprotection is under active investigation in several laboratories (20). There is evidence that mitoKATP channels are involved in the mechanism of IPC (35,36). Furthermore, the mitoKATP channel has also been suggested as a possible end effector in the mechanism of DP afforded by adenosine A1-receptor activation (21), δ1-opioid receptor stimulation (37), or heat stress (26). Mei et al. (23) showed that the delayed cardioprotective effects of MLA, which contains the same moiety of lipid A as LPS, are abolished by blocking the mitoKATP channel with 5-HD. These findings encouraged us to investigate whether DP afforded by LTA from S. aureus or LTA from B. subtilis is also mediated by the mitoKATP channel.
It is surprising that late administration of 5-HD did not affect the delayed cardioprotective effects of LTA from either strain of bacteria. The dose (5 mg/kg i.v.) and administration regimen (10 min before LAD ischemia-reperfusion) of 5-HD used in this study was previously shown to abolish the cardioprotective effects of different prostanoids in identical models of coronary artery occlusion-reperfusion (38–40). Similarly, others have shown that 5-HD (5–10 mg/kg i.v.) or glibenclamide administered (5–20 min) before coronary artery occlusion blocks DP afforded by a range of other treatments (21,23,41,42). For these data and other studies, many investigators have used 5-HD only as a selective blocker of mitoKATP channels. Although our laboratory has extensive experience with 5-HD, we cannot exclude in this study that 5-HD did not reach the mitoKATP channel in vivo for some reasons. In conclusion, the inability of 5-HD to abolish DP afforded by LTA from either S. aureus or B. subtilis does not support a role for the mitoKATP channel as the end effector involved.
ACKNOWLEDGMENTS
KZ is supported by the Deutsche Herzstiftung.
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