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Am J Physiol Heart Circ Physiol 278: H1717-H1724, 2000;
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Vol. 278, Issue 5, H1717-H1724, May 2000

A new method of long-term preventive cardioprotection using Lactobacillus

Tatyana Oxman1, Michal Shapira2, Adriana Diver1, Rodica Klein1, Natalie Avazov1, and Babeth Rabinowitz1

1 Heart Institute, Chaim Sheba Medical Center, Tel Hashomer, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv 69978; and 2 Department of Life Sciences, Ben Gurion University, Beer Sheba, Israel 84105


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Potential long-term cardioprotection was investigated in an extensive experimental study. Lactobacillus cultivation components (LCC) were administered intravenously in anesthetized rats 1, 7, and 21 days before global ischemia (GI). GI was produced by full stop flow in isolated Langendorff-perfused hearts for 20 min and was followed by reperfusion. Control animals were injected with saline. LCC reduced reperfusion tachyarrhythmia significantly and improved functional recovery of the ischemized rat heart. These beneficial effects were associated with reduction of release of norepinephrine (NE) and prostacyclin at the first minute of reperfusion, activation of myocardial catalase, and overexpression of 70-kDa heat stress protein (HSP-70) at ischemia and reperfusion (P < 0.05). This cardioprotection was documented up to 21 days after a single injection of LCC. Thus Lactobacillus cultivation components are new nontoxic materials that produce marked long-term cardioprotection against ischemia-reperfusion damage. This effect is attributed to an activation of the cellular defense system, manifested by activation of the antioxidant pathway and by expression of protective proteins. NE is involved in this process, and the data also suggest a role for prostacyclin in this model of cardioprotection. The potential of LCC and related compounds working through similar mechanisms in the prevention and therapy of various ischemic heart syndromes should be explored.

heat stress proteins


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

CARDIOPROTECTION BY ADAPTATION of the heart to ischemia is a complex and multifaceted phenomenon with important potential clinical applications. Ischemic preconditioning is a form of immediate cardioprotection, obtained by episodes of short-term ischemia and reperfusion before a long ischemic period. The "second window of protection" is a long-term process that may occur several hours after an ischemic event and may last up to several days (9, 21, 23). Adaptation to ischemia, perhaps for longer periods of time, can also be obtained by a pharmacological approach (4, 11, 28). Previous reports suggest that heat shock proteins (HSPs) (10, 13, 14, 20, 25, 29, 37) and proteins with anti-oxidative activity are involved in myocardial adaptation (16, 17, 22, 39). Hormone-mediated signaling mechanisms may also be involved (3, 15, 32, 35).

As early as 1936, Weichardt (34) observed that "the administration of certain nonspecific damaging substances leads to the production of metabolites, which increase the resistance of the organism against various diseases." Increased myocardial tolerance to a subsequent challenge against ischemia and reperfusion by sublethal doses of gram-negative bacterial endotoxin (lipopolysaccharides; LPS) is well known (6, 28, 33). However, the toxic nature of endotoxin has precluded its clinical application. The use of an endotoxin analog with reduced toxicity, the 4'-monophosphoryl derivative of lipid A (MLA), opened up new possibilities for studying protective mechanisms. MLA administration, 24 h before global or regional ischemia followed by reperfusion, enhanced functional recovery of the heart, reduced infarct size, and decreased the incidence of ventricular tachycardia (VT) as well as ventricular fibrillation (VF) (11, 36). These protective effects were accompanied by activation of an antioxidant pathway (4, 39). The involvement of HSPs (also known as heat stress proteins) in MLA-induced myocardial protection is in dispute, with some reports ruling it out (4, 38) and others supporting their increased expression in response to MLA administration (6a, 24). Therefore, the mechanism by which either MLA or endotoxin exerts its cardioprotective effect remains unclear.

Despite the reduced toxicity of MLA, such agents are always restricted to a limited dose due to the danger of toxic effects on administration of increased doses. To develop a new direction of nontoxic bacterial-derived protective agents, the myocardial protective effects of a Lactobacillus preparation were examined. Lactobacillus is not toxic to cells and organisms and could therefore be utilized for the development of new drugs that have no toxic effects.

We hypothesized that increased myocardial tolerance to ischemia-reperfusion damage, similar to that demonstrated with gram-negative bacteria (endotoxin), might be obtained by using different bacterial strains capable of enhancing nonspecific resistance. Some properties of Lactobacillus associated with previously reported studies (7, 8) are that 1) Lactobacillus is generally recognized as a safe organism, based on the fact that it does not contain LPS and lipid A in its cell walls, and 2) it possesses the ability to stimulate the host's nonspecific immunity (adjuvanticity). These properties led us to consider these gram-positive bacteria.

The goal of this study was to investigate the potential of a nontoxic product of Lactobacillus (gram-positive bacteria) to induce long-term cardioprotection against ischemia-reperfusion injury in rat hearts. In addition, the study was designed to determine whether this protection is associated with the activation of a cellular defense mechanism including myocardial catalase activity and 70-kDa HSP (HSP-70) expression.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and Treatment

Male Sprague-Dawley rats (body wt 250-350 g) were used. The investigation conformed to the Guide for the Care and Use of Laboratory Animals, published by the National Research Council.

Lactobacillus cultivation components (LCC) were obtained by fermentation and lyophilization of Lactobacillus bulgaricus-51. The production of biomass was carried out by the Minerva-Otto Meyerhof Biotechnological Laboratories (Technion, Haifa, Israel). A previously determined nontoxic dose of LCC (45 mg/kg) was used.

To examine whether LCC administration induced prolonged cardioprotection, anesthetized rats (pentobarbital sodium, 5 mg/100 g ip) were treated with LCC. Lyophilized LCC, freshly diluted in 1 ml of normal saline, was administered intravenously. Control animals were injected intravenously with 1 ml of normal saline. Rats were anesthetized and killed at 1, 7, or 21 days after injection, and isolated hearts were subjected to global ischemia (GI) and reperfusion.

Measurement of Rectal Temperature and Hemodynamics in Anesthetized Rats

The effect of LCC on rectal temperature was measured before the intravenous injection and after LCC administration for a period of 30 min. The right femoral artery was cannulated for direct assessment of arterial blood pressure and heart rate. After 20 min of stabilization, LCC (or saline) was administered intravenously. Arterial blood pressure and heart rate were continuously recorded with a pressure transducer (SENSO NOR-840). The hemodynamic parameters were recorded on a polygraph (Astro-Med 8800).

Isolated Heart Perfusion and Assessment of Cardiac Function and Tachyarrhythmia

Rats received 1,000 units of heparin and 5 mg/100 g pentobarbital sodium intraperitoneally. The hearts were rapidly excised, arrested in ice-cold heparinized saline, and mounted on the Langendorff isolated-heart apparatus. A modified Krebs-Henseleit solution consisting of 118 mM NaCl, 25 mM NaHCO3, 11.1 mM glucose, 4.9 mM KCl, 2.7 mM CaCl2, 1.2 mM MgSO4, and 0.5 mM Na-EDTA was equilibrated with 95% O2 and 5% CO2. The whole system was heated to 37°C by means of water jacketing. The heart was suspended in a sealed chamber containing a silicone funnel for collection and volumetric measurement of the coronary flow. A 15-min stabilization period was performed in each group before ischemia of the heart.

For investigation of tachyarrhythmia during reperfusion, the perfusate column height was kept at 75-80 cmH2O, producing a perfusion pressure of ~50 mmHg. A stainless steel electrode was inserted into the right ventricular epicardium for bipolar electrogram recording relative to the aortic cannula. Hearts were allowed to beat spontaneously during the entire experiment. A three-way stopcock above the aortic root was turned to create GI or reperfusion.

Analyses of VT and VF were carried out in accordance with the Lambeth conventions. Distinguishing between VT and VF on a "per animal" basis was difficult in this model because of numerous intermediate grades of tachycardia and a tendency to convert from one type to another. Therefore, we defined VT or VF of at least 5 beats/min as ventricular tachyarrhythmia. Tachyarrhythmia (TA), persisting for a similar period of time, up to the end of reperfusion, was referred to as sustained TA.

To study functional recovery of the hearts, an H2O-filled balloon was inserted through the atrium to the left ventricle and adjusted to an end-diastolic pressure of 5 mmHg. The balloon was connected via a polyethylene catheter to a pressure transducer. Thereafter, the balloon volume was not changed. Perfusion pressure was constant at 60 mmHg. The perfused column height was kept at 100 cmH2O. Left ventricular systolic pressure (LVSP) and left ventricular end-diastolic pressure (LVEDP) were continuously recorded with a pressure amplifier and direct differentiation to obtain ±dP/dt (where P is pressure and t is time). Left ventricular developed pressure (LVDP) was calculated from LVSP minus end-diastolic pressure. Rate-pressure product (RPP) was calculated as LVDP × heart rate. Pacing wires were fixed to the right auricle and to the aorta, and the hearts were paced at 6 Hz. Paced hearts that did not produce 90 ± 10 mmHg LVDP during stabilizing perfusion were discarded. During GI the hearts were held in a closed chamber heated to 37°C. Pacing was resumed 3 min after the start of reperfusion.

Stabilization of perfusion for 15 min was performed in all experiments and was followed by 20 min of GI and 15 min of reperfusion in examinations of reperfusion tachyarrhythmia incidence and 30 min of reperfusion in investigations of cardiac function.

Biochemical Assays of the Coronary Effluent

Samples from the coronary effluent were collected after 15 min of stabilization, just before ischemia (baseline), and at 1-3 min of reperfusion. The samples were kept at -20°C until the assay.

Production of prostacyclin (PGI2) was assessed by measurement of its stable metabolite 6-keto-PGF1alpha , with the use of a DuPont 3H-labeled radioimmunoassay kit (27). Norepinephrine (NE) was determined by high-pressure liquid chromatography with electrochemical detection (12). The results were expressed as picograms per minute per gram wet weight.

Tissue Lactate and ATP Determinations

Hearts obtained just before GI, after 20 min of GI, or after 15 min of reperfusion were rapidly frozen in liquid nitrogen, weighed, and kept at -80°C until the assay. Commercially available kits (Sigma Diagnostics) were used for the enzymatic determination (1, 19). The results were expressed in micromoles per gram of tissue.

Determination of Myocardial Catalase Activity

Just before GI, after GI, and after 15 min of reperfusion, the hearts were rapidly weighed and extracted in the presence of 0.17 mM ethanol and 1% Triton X-100 in a hypotonic phosphate buffer, pH 7.0. An ultraviolet spectrophotometer (240 µm) method was used (2). The catalase activity was expressed in units per gram wet weight per minute.

Protein Isolation and Western Blotting

Whole hearts (n = 4 in each specified treatment) were excised and frozen in liquid nitrogen, weighed, and stored at -80°C before protein isolation. The hearts were homogenized (100 mg/ml) by a Polytron homogenizer in SDS-PAGE sample buffer in the presence of proteinase inhibitors. Protein concentration was determined by the Pierce bicinchoninic acid (BCA) protein assay. Equal protein quantities, pretested to be within the linear range of enhanced chemiluminescence (ECL) detection and Coomassie blue staining of the actin band, were separated on two parallel 10% SDS-polyacrylamide gels (17a). One of the gels was blotted onto a nitrocellulose membrane and further processed for Western analysis, and the other gel was subjected to Coomassie staining to visualize the immobilized protein bands.

The blots were then incubated with a monoclonal antibody raised against HSP-70, recognizing both constitutive and inducible forms of the protein (1:5,000; Sigma), with a conjugate of horseradish peroxidase and affinity-purified goat anti-mouse antibodies (1:5,000; Jackson Laboratories) and further developed by ECL detection (Amersham).

Experimental and control samples from each time point were analyzed on the same gel, and the relative levels of HSP-70 and actin in the different samples were determined by densitometry (Molecular Dynamics densitometer model PD, using ImageQuant software). HSP-70 expression was normalized to the actin band on the Coomassie-stained gels to adjust for slight variations in the protein loading between samples. Changes in the level of HSP-70 were presented as the ratio between expression in the LCC-treated animals and in the saline-injected control animals, following similar myocardial procedures.

Statistical Analysis

Categorical data, i.e., TA incidence, were compared with Fisher's test. Other results were compared by ANOVA with Duncan intergroup comparisons of repeated measures as required. Statistical significance was accepted at two-sided P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of LCC Administration on Rectal Temperature and Hemodynamics in Anesthetized Rats

No marked changes in rectal temperature were observed in the anesthetized rats treated either with saline or with LCC over a period of 30 min after the intravenous bolus (35 ± 1°C). An intravenous bolus of saline caused a rapid and marked increase in systolic aortic pressure (SAP; Table 1). The maximal rise in SAP (15 ± 4.6% above baseline) was observed during the injection of saline, and it returned to the baseline level 5 min later. There were no significant changes in heart rate during and after injection of saline. An intravenous bolus of LCC caused a rapid and marked decrease in SAP. The maximal reduction in SAP was observed during the administration of LCC (40 ± 5.3% below baseline). SAP recovered to the baseline within 30 min after injection. The heart rate decreased during LCC administration (P < 0.05) and returned to baseline levels after 5 min.

                              
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  		Table 1.   Effects of LCC on hemodynamics in anesthetized rats

Effects of LCC Pretreatment on Reperfusion Arrhythmias

TAs developed at 30-90 s of reperfusion and were self-limited. The hearts resumed normal sinus rhythm after the episodes of TA terminated and remained in that rhythm until the end of the experimental protocol. Table 2 compares the incidence and duration of TA in the various experimental groups.

                              
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  		Table 2.   Effect of LCC pretreatment on reperfusion tachyarrhythmia

The frequency of TA was significantly different between control (saline pretreated) and LCC-pretreated hearts. Seventy-three percent of the hearts taken from the control rats had TA, whereas in the LCC-pretreated rats, only 19, 18, and 25% of the hearts showed TA when excised 1, 7, and 21 days after LCC injection, respectively. Thus LCC pretreatment induced a significant antiarrhythmic effect for a period of 1-21 days.

Effects of LCC Pretreatment on Cardiac Function During Reperfusion

Coronary flow. As depicted in Table 3, the coronary flow measured at the baseline after 15 min of stabilizing perfusion was similar in all the hearts. During reperfusion, the coronary flow significantly decreased in all groups, with no marked influence of LCC administration. Coronary flow in reperfused hearts decreased less in both groups 1 day after pretreatment with either saline or LCC compared with data from days 7 and 21. No marked differences were observed 7 and 21 days after pretreatment with either saline or LCC. The enhancement of coronary flow 1 day after any of the pretreatments could be due to anesthesia and intravenous injections 1 day before ischemia.

                              
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  		Table 3.   Coronary flow after LCC pretreatment

Cardiac function. Ischemia produced pronounced changes in cardiac function during reperfusion: decrease of RPP, LVDP, and ±dP/dt and increase of LVEDP (P < 0.05 vs. preischemic level; Table 4). At day 1 after injection of either saline or LCC, the studied parameters of cardiac function altered only minimally in comparison with the preischemic level and with the changes at days 7 and 21. It would seem, therefore, that anesthesia and intravenous injection provoke a stress effect, which can affect the preconditioning after ischemia over the first 24 h.

                              
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  		Table 4.   Effects of LCC pretreatment on cardiac function at reperfusion after GI

LCC pretreatment 7 and 21 days before ischemia improved functional recovery of the heart in reperfusion after 20 min of GI. RPP and LVDP, 7 and 21 days after LCC injection, were significantly increased at all points examined in comparison with corresponding control hearts (Fig. 1). Hearts isolated 7 and 21 days after LCC treatment demonstrated a significantly attenuated increase in LVEDP during reperfusion. LCC pretreatment 7 and 21 days before prolonged ischemia significantly increased the contractility of the heart during reperfusion compared with the untreated hearts (P < 0.05; Fig. 2).


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  		Fig. 1.   Left ventricular developed pressure during reperfusion after 20 min of global ischemia (GI). Each bar represents mean ± SE of measurements obtained in 10 experiments at a given time point. Data are expressed as percentage of preischemic values. LCC, Lactobacillus cultivation component; A, 15-min reperfusion; B, 30-min reperfusion. * P < 0.05 vs. control.



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  		Fig. 2.   Contractility of isolated rat heart during reperfusion after 20 min of GI. Each bar represents mean ± SE of measurements in 10 experiments at a given time point. Data are expressed as percentage of preischemic values. a: dP/dtmax. b: dP/dtmin. a and b: P is pressure, and t is time. A, 15-min reperfusion; B, 30-min reperfusion. * P < 0.05.

Effects of LCC Pretreatment on the Release of NE and PGI2

LCC pretreatment did not induce marked changes in the release at the preischemic level of either NE or PGI2. The control hearts, submitted to 20 min of GI, released increased amounts of NE and PGI2 at 1-3 min of reperfusion. LCC pretreatment resulted in an increase of NE release at reperfusion, but this rise was significantly lower compared with the control hearts (Table 5). During reperfusion, PGI2 release was significantly lower compared with the control and did not differ from the preischemic level. Thus LCC pretreatment induced a reduction in the release of NE and PGI2 at reperfusion after GI in rat hearts over a period of 21 days.

                              
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  		Table 5.   Effects of LCC pretreatment on release of NE and PGI2 in reperfusion after 20-min GI

Effects of LCC Pretreatment on Tissue Lactate and ATP

LCC injection had no marked influence on the level of ATP and lactate compared with the baseline control (Table 6). Ischemia sharply increased the level of lactate and decreased the quantity of ATP in the myocardial tissue. In LCC-treated hearts, the level of intracellular ATP increased by 139% at days 1 and 7, whereas the level of lactate was slightly decreased (to 93%) at both day 1 and day 7 (P < 0.05). We did not find significant changes in the lactate and ATP content during reperfusion.

                              
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  		Table 6.   Lactate and ATP in heart tissue in GI and reperfusion after LCC pretreatment

Effects of LCC Pretreatment on the Myocardial Catalase Activity

The baseline catalase activity increased significantly only at day 1 after LCC administration. However, no changes were observed after 7 and 21 days of the LCC treatment compared with the corresponding control hearts.

GI reduced the catalase activity in the control hearts to ~45% compared with baseline. However, despite the decrease, if LCC was administered 7 and 21 days before GI, catalase activity was maintained at significantly higher levels compared with the saline-treated hearts (Table 7), and at reperfusion, catalase activity was higher in all LCC-pretreated hearts compared with the corresponding controls. Thus LCC pretreatment enhanced myocardial catalase activity in GI and in reperfusion during 21 days.

                              
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  		Table 7.   Effect of LCC pretreatment on catalase activity of heart

Effects of LCC Pretreatment on HSP-70 Expression

The results of Western blotting of HSP-70 represent a typical experiment, which was included in the statistical analysis (Fig. 3). GI evoked expression of HSP-70 in both control and LCC-pretreated hearts, as expected. However, after 20 min of GI, expression of HSP-70 increased in LCC-pretreated hearts by 40, 73, and 32% at 1, 7, and 21 days, respectively, compared with the control hearts. The ischemia-induced expression of HSP-70 compared with baseline was significantly higher in the LCC-pretreated hearts (131% at 1 day and 202% at 7 days) compared with the control hearts (Table 8).


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  		Fig. 3.   Western analysis of 70-kDa heat stress protein (HSP-70) expression in perfused rat hearts after LCC administration. Rat heart homogenates containing equal protein quantities (bicinchoninic acid tested) were separated over duplicate 10% SDS-polyacrylamide gels. One of the gels was blotted onto a nitrocellulose membrane and reacted with anti-HSP-70 antibodies, and the other was stained by Coomassie to ensure equal protein loads. HSP-70 binding was detected by enhanced chemiluminescence and is shown in each top panel. Staining of actin band is shown at corresponding bottom panels. HSP-70 levels were monitored before (baseline) and during ischemia and reperfusion at 1, 7, and 21 days after administration of LCC. Control (C) animals were injected with saline and subjected to similar treatments.


                              
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  		Table 8.   Effects of LCC pretreatment on HSP-70 expression at GI

The data obtained at reperfusion also showed a significant overexpression of HSP-70 in the LCC-pretreated hearts compared with the control hearts: 71, 53, and 40%, respectively (Table 9). It is important to note that LCC pretreatment did not evoke overexpression of HSP-70 in the perfused hearts at baseline. Thus LCC pretreatment induced an HSP-70 overexpression that could be observed up to 21 days.

                              
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  		Table 9.   Effect of LCC pretreatment on HSP-70 expression at GI and reperfusion in rat hearts


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study shows that a preparation of Lactobacillus induces long-term cardioprotection against deleterious effects of cardiac ischemia. To the best of our knowledge, this is the first study to demonstrate that adaptation to ischemia-reperfusion damage can be obtained with gram-positive bacteria. Cardiac protection induced by LCC is not immediate; it appears first at 24 h after administration and is observed for up to 21 days.

Previous investigations documented cardioprotection for a maximum of 24-48 h with the use of bacterial endotoxin or its derivative, MLA (11, 33, 36). The cardioprotective effects induced by these agents were reduction in ventricular arrhythmia, improvement of functional recovery, and decrease of infarct size. The LCC-induced cardioprotective effects demonstrated by us were a marked reduction of ventricular arrhythmia and an improved recovery of cardiac function for up to 21 days.

This delayed cardioprotection is associated with increased myocardial ATP and a slight decrease in lactate during ischemia, which may play a role in the improved functional recovery of the heart during reperfusion. LCC pretreatment reduced the release in both NE and prostacyclin at reperfusion. The reduction of NE release has been previously reported in isolated rat hearts subjected to ischemic preconditioning (32). The significant decrease of NE release in LCC-treated hearts suggests that this substance is involved in LCC-induced cardioprotection. It is well known that prostacyclin is a tissue protective factor in myocardial ischemia (35) that has a beneficial influence on the reperfused ischemic myocardium (15, 30). The significant reduction of prostacyclin release during reperfusion after LCC administration suggests a relationship between prostacyclin and cardioprotection in this model.

Stress-induced proteins have been implicated with the protection of cells and tissue against the deleterious effects of stress, including ischemia (26). The relatively long-term cardioprotection obtained by LCC is associated with activation of myocardial catalase and HSP-70 overexpression during ischemia and reperfusion. HSP-70 overexpression is not observed after LCC administration, but ischemia of LCC-treated hearts lead to a higher expression of this protein. A parallel phenomenon was observed with exposure of HeLa cells to mild organic acids that function as anti-inflammatory drugs, namely, indomethacin and salicylate. Under these conditions, expression of HSP-70 was not induced; however, when cells treated with salicylate were exposed to temperature stress, the threshold for heat shock transcription factor (HSF) activation was reduced (18). Molecular studies aimed to decipher the mechanism that underlies this observation revealed that pretreatment with indomethacin triggered the binding of HSF to the corresponding heat shock element (HSE) in the promoter region, but this binding did not result in transcriptional activation of the HSP-70 gene. Transcription of HSP-70 was not induced, since the bound transcription factor was not subject to the inducible phosphorylation that precedes transcriptional activation. However, during a subsequent heat shock, the bound HSF became phosphorylated at a lower threshold, leading to an increase in the cytoprotective effect from the temperature-induced damages, which lasted for a longer period (18). Thus the drug treatment by itself did not induce the heat shock response, but it altered the expression of HSPs during a subsequent temperature stress, conferring an improved cytoprotective effect. We suggest that an analogous mechanism could explain the long-term protection exerted by LCC administration, namely, that in LCC-treated animals, the HSF may bind to the HSE in its nonphosphorylated form. Thus, on ischemia, the bound HSF could become phosphorylated, driving expression of HSP-70 to a higher level than that observed in nontreated hearts. Further investigation is required to prove this hypothesis and to identify the exact components of the Lactobacillus preparation that are involved in triggering a stronger response of HSP-70 expression and antioxidant activity, which are not yet known. However, we suggest that the long-term effects could be of a similar nature to those induced by salicylates and anti-inflammatory drugs.

The activation of catalase observed by us in the LCC-treated hearts is similar to the data reported by other investigators who used endotoxin (4, 6, 38). However, the protective effects induced by endotoxin and MLA did not always result in an increased expression of HSP-70 (4, 38). We therefore assumed that the protective pathways exercised by LCC and endotoxin or MLA administration are not the same, which may be explained by a difference in the structure of the cell walls between gram-positive and gram-negative bacteria. The cell wall of gram-negative bacteria contains LPS and lipoproteins, including lipid A, which are not present in the cell walls of gram-positive bacteria. In the latter, the majority of the cell wall consists of peptidoglycan, which is present in a smaller amount in gram-negative bacteria (8). The toxicity of gram-negative bacteria stems from the LPS and lipoprotein fractions, which are absent in gram-positive bacteria.

The mechanisms of LCC-induced cardioprotection are not yet entirely clear, and the specific component in the Lactobacillus preparation that confers long-term cardioprotection is yet to be identified. As with endotoxin and MLA, it is possible that LCC administration elicits production of cytokines and induces inducible nitric oxide synthase expression (5, 40) involving cellular mechanisms of protection, such as activation of the antioxidant pathway and HSP-70 expression. However, given the high toxicity of gram negative-derived components, the development of a gram-positive nontoxic treatment is of great advantage. Thus certain recent reports indicate that Lactobacillus can serve as a safe oral vaccine to induce host-nonspecific immunity (7). It is of the utmost importance that the gram positive-based treatment be a safe preparation suitable for long-term use.

In conclusion, this work is the first to demonstrate the ability of a nontoxic preparation from a gram-positive bacteria to induce long-term cardioprotection against deleterious effects of prolonged ischemia. We suggest that this cardioprotection is part of an enhanced nonspecific resistance of the body to different forms of stress, including ischemia and reperfusion. Although the precise mechanism of LCC-induced adaptation is unknown, our data suggest that this cardioprotection is attributed to an activation of the cellular defense system, manifested by activation of the antioxidant pathway and expression of protective proteins. The potential of LCC and related compounds, working through similar mechanisms in the prevention and therapy of various ischemic heart syndromes, deserves to be explored.


    ACKNOWLEDGEMENTS

We are grateful to Penina Reichenberg and Rivka Rosenberg for invaluable administrative and editorial assistance.


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: Babeth Rabinowitz, Heart Institute, Sheba Medical Center, 52621 Tel Hashomer, Israel (E-mail address: babethr{at}post.tau.ac.il).

Received 4 February 1999; accepted in final form 15 December 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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6.   Brown, JM, Grosso MA, Tarada GJ, Whitman GY, Banerjee A, White CW, Marken AM, and Repine YF. Endotoxin pretreatment increases endogenous myocardial catalase activity and decreases ischemia-reperfusion injury of isolated rat heart. Proc Natl Acad Sci USA 86: 2516-2520, 1989[Abstract/Free Full Text].

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8.   Cockayne, A. Bacterial cell walls. In: Encyclopedia of Immunology, edited by Roith IM, and Delves PJ.. London: Academic, 1992, p. 216-229.

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Am J Physiol Heart Circ Physiol 278(5):H1717-H1724
0363-6135/00 $5.00 Copyright © 2000 the American Physiological Society



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