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Cardiovascular Research 2006 72(1):134-142; doi:10.1016/j.cardiores.2006.06.029
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Copyright © 2006, European Society of Cardiology

Cardiac ICAM-1 mediates leukocyte-dependent decreased ventricular contractility in endotoxemic mice

Ehsan Y. Davani, John H. Boyd, Delbert R. Dorscheid, Yingjing Wang, Anna Meredith, Edmond Chau, Gurpreet K. Singhera and Keith R. Walley*

Critical Care Research Laboratories, St. Paul's Hospital, University of British Columbia, 1081 Burrard Street, Vancouver, BC, Canada V6Z 1Y6

* Corresponding author. Tel.: +1 604 806 8136; fax: +1 604 806 8351. Email address: kwalley{at}mrl.ubc.ca

Received 28 March 2006; revised 6 June 2006; accepted 23 June 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Binding of ICAM-1 expressed on cardiomyocytes decreases cardiomyocyte contractility in vitro by altering the intracellular Ca2+ transient. We tested the hypothesis that signaling via ICAM-1 contributes to decreased left ventricular contractility in an in vivo model of systemic inflammation.

Methods: C57B6 wild-type mice and ICAM-1 knock-out mice were treated with intraperitoneal lipopolysaccharide (LPS) then left ventricular contractility was measured 6 h later using a volume-conductance micromanometer catheter. We repeated this experiment in chimeric mice lacking ICAM-1 expression in bone marrow-derived cells (M–) and/or lacking ICAM-1 expression in the heart and other tissues (H–).

Results: In C57B6 wild-type mice LPS injection significantly increased cardiac ICAM-1 expression and decreased in vivo measures of left ventricular contractility (end-systolic elastance, Ees decreased 58±4%, p<0.05, [dP/dtmax]/EDV decreased 60±6%, p<0.05). Cyclophosphamide pretreatment to decrease leukocyte count prevented the LPS-induced decrease in contractility. In ICAM-1 knock-out mice LPS did not decrease any measure of contractility. LPS did not decrease left ventricular contractility in M+/H– mice but decreased contractility in M+/H+ and M–/H+ mice to the same extent as in C57B6 wild-type mice implicating the importance of cardiac ICAM-1.

Conclusions: We conclude that signaling via cardiac ICAM-1 is necessary to mediate leukocyte-dependent decreases of left ventricular contractility in endotoxemic mice.

KEYWORDS Contractile function; Infection/inflammation; Leukocytes; Sepsis


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Sepsis is responsible for more than 200,000 deaths annually in North America [1]. Myocardial dysfunction, caused by the systemic inflammatory response of sepsis, contributes to multi-organ dysfunction and death in these patients [2]. When the degree of myocardial dysfunction is substantial, cardiac output cannot be increased sufficiently to maintain an adequate systemic arterial pressure in the face of the decreased systemic vascular resistance of septic shock. Indeed high versus low cardiac output during septic shock distinguishes survivors from non-survivors [3]. Leukocytes are a component of the systemic inflammatory response of sepsis and have been implicated in contributing to myocardial dysfunction in experimental models both in vivo [4,5] and in vitro [6–9].

Leukocytes can decrease isolated cardiomyocyte contractility in vitro by numerous mechanisms, the nature of which depends to some degree on the nature of the inflammatory stimuli. When polymorphonuclear cells are exposed to formulated peptides of bacterial origins, {alpha}4 integrins mediate an oxidant induced reduction in contractility on cocultured cardiac myocytes [8,10]. Various inflammatory stimuli up-regulate CD18 on leukocytes, causing specific binding to ICAM-1 expressed on the cardiomyocytes [7–9]. In addition, even non-activated leukocytes or ICAM-1 cross-linking antibodies can decrease contractility by binding ICAM-1 expressed on cardiomyocytes [7,9]. ICAM-1 binding then mediates decreased cardiomyocyte contractility by signaling via the cortical actin cytoskeleton, which leads to increased heterogeneity of intracellular Ca2+ release and decreased cardiomyocyte contractility [9]. Whether ICAM-1 binding plays an important role in the development of myocardial dysfunction in vivo has not been demonstrated.

Accordingly, we first determined the extent of increased cardiac ICAM-1 expression using a lipopolysaccharide (LPS) injection model of systemic inflammation in mice. Next, we tested the hypothesis that myocardial dysfunction occurs in this model and, further, we determined whether the degree of myocardial dysfunction is prevented by decreasing the peripheral blood leukocyte count. We then determined whether ICAM-1 expression is also necessary for the development of myocardial dysfunction in this model, using ICAM-1 knock-out mice. Finally, to determine if cardiac ICAM-1 expression is necessary for myocardial dysfunction (versus ICAM-1 expressed on bone marrow-derived cells), we developed chimeric mouse models lacking ICAM-1 expression in bone marrow-derived cells and/or lacking ICAM-1 expression in the heart and other tissues. We found that activation of cardiac ICAM-1 plays a key role in mediating decreased left ventricular contractility in this whole animal model of systemic inflammation.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
This study was approved by the University of British Columbia Animal Care Committee and adheres to Canadian and U.S.A. National Institutes of Health guidelines for animal care.

2.1. Experimental preparation
Mice, C57B6 wild-type and C57B6/J-ICAMtm1-jcgr knock-out (ICAM-1 knock-out, Jackson Laboratory, Bar Harbor, ME) weighing 25–30 g, were given intraperitoneal injection of purified lipopolysaccharide (LPS, E. coli strain 01 II: B4, Sigma, St. Louis, MO) 40 mg/kg or the same volume of normal saline. Six hours after LPS injection, mice were anesthetized using ketamine (75 mg/kg) and xylazine (10 mg/kg); total volume 0.2 mL, injected subcutaneously. Anesthesia effectiveness was confirmed throughout the procedure. Tracheal intubation was performed through a 1 cm midline neck incision and mice were ventilated (Mouse Ventilator model 687, Harvard Instruments, Holiston, MA) at 120 breaths/min with a 200 µL tidal volume. A substernal transverse incision was made to expose the apical portion of the heart and inferior vena cava. The left ventricle was punctured using a 27 gauge needle approximately 1 mm lateral to the interventricular septum. Then a number 2 French volume-conductance micromanometer catheter (Mikro-tip SPR-838, Millar Instruments Inc., Houston, TX) was inserted into the left ventricle. Correct placement of the catheter was determined by checking pressure–volume loops for vertical isovolumic phase traces and characteristic diastolic and systolic trajectories. Pressure–volume data were recorded at steady state and the inferior vena cava was occluded for 3–5 s while pressure–volume data were again recorded. The heart was excised and frozen (isopentane in liquid nitrogen) or fixed (10% formalin) for further histopathology and morphometry measurements.

2.2. Left ventricular contractility and cardiac function
Left ventricular contractility and other measures of ventricular function were determined from pressure–volume measurements using Pressure–Volume Analysis software (PVAN 2.9, Millar Instruments Inc., Houston, TX). Six to ten pressure–volume loops during a vena-cava occlusion were sampled and used to measure end-systolic elastance (Ees), which is the slope of the end-systolic pressure–volume relationship (Fig. 1). Ees is an ejection phase measure of left ventricular contractility which, among many others, is least sensitive to changes in preload and afterload [11] anticipated to occur in this model of sepsis. The volume axis intercept, Vd, was considered zero volume for the steady-state measurement of end-diastolic volume (EDV) and end-systolic volume (ESV). Pressure–volume loops measured during steady-state conditions were used to measure the maximum rate of change of intraventricular pressure during isovolumic systole divided by EDV, [dP/dtmax]/EDV, which is a sensitive isovolumic phase measure of left ventricular contractility [12]. Steady-state pressure–volume loops were also used to calculate ejection fraction as the difference between EDV and ESV divided by EDV, which is a further measure of cardiac function. End-systolic pressure during steady state was used as a measure of systemic arterial pressure afterload.


Figure 1
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  		Fig. 1 Pressure–volume measurements of multiple heart beats during a vena-caval occlusion are illustrated. A best fit line to the end-systolic points of these beats (heavy solid line) is the end-systolic pressure–volume relationship. The slope of the end-systolic pressure–volume relationship is end-systolic elastance, Ees; a preload and afterload independent measure of left ventricular contractility.

 
2.3. The role of leukocytes
To determine whether leukocytes contributed to decreased left ventricular contractility and function in this animal model we increased leukocyte count by pretreating C57B6 wild-type mice with GCSF and, alternatively, we decreased leukocyte count by pretreating C57B6 wild-type mice with cyclophosphamide. Mice were then injected with LPS and left ventricular contractility and function were measured as above and compared to C57B6 wild-type mice treated with LPS.

To increase leukocyte count, mice received GCSF (Filgrastim 300 µg/ml vial, Neupogen) 120 µg/kg subcutaneously twice daily for four days and were then rested for 3 days. This time course was determined from preliminary experiments where we measured the ratio of CD11b expression to total actin content as an indicator of bone marrow cell maturation. Compared to non-treated controls we found a significant decrease in this ratio immediately after the 4-day GCSF treatment. Following 3 days of rest, the ratio of CD11b expression to total actin content returned towards normal in the GCSF-treated mice indicating that 3 days of rest after GCSF injection was necessary to approximate normal bone marrow cell maturation.

To decrease leukocyte count, mice received cyclophosphamide (Bristol Laboratories, Montreal, Canada) 150 mg/kg intraperitoneal daily for 2 days and were then rested for 3 days. This time course was determined from preliminary experiments and the literature which indicated that the nadir of the peripheral blood leukocyte count occurred 3 days after the last cyclophosphamide injection [13–15].

To confirm the effect of GCSF and cyclophosphamide, we measured total leukocyte count and polymorphonuclear leukocyte (neutrophil) count in peripheral blood and in bone marrow using a Cell-Dyn 3700 counter (Abbott Diagnostic Division, Abbott, IL).

2.4. Chimeric models
We reasoned that any difference in LPS effect found in ICAM-1 knock-out mice could either be due to loss of cardiac ICAM-1 expression or due to loss of ICAM-1 expression on blood borne bone marrow-derived cells transiting the heart. To distinguish between these two possibilities, and thereby specifically implicate cardiac ICAM-1 expression, we designed three different chimeric mouse models; ICAM-1 expressed on marrow-derived cells and on heart cells (M+/H+), ICAM-1 expressed on marrow-derived cells but not on heart cells (M+/H–), and ICAM-1 not expressed on marrow-derived cells but expressed on heart cells (M–/H+).

We developed these chimeric mice as follows. To increase the number of progenitor cells in donors, C57B6 wild-type and ICAM-1 knock-out mice were first pretreated with GCSF as described above. Bone marrow from femurs and tibias of 8 to 10 GCSF-treated mice (donors) was extracted and separated in PBS plus ACD 10% (C3821 Acid Citrate–Dextrose solution, Sigma), washed 4 times in PBS plus ACD and filtered. After a final wash in PBS, cells were suspended in normal saline. In this bone marrow concentrate the total leukocyte count was 12.2±1.2x109/L, the fraction of granulocytes was 94.8±0.3%, and the fraction of lymphocytes was 3.5±0.1%. To suppress inflammatory cell progenitors in recipient mice, C57B6 wild-type and ICAM-1 knock-out mice were first pretreated with cyclophosphamide as described above.

Bone marrow cells concentrate (300 µL) from the donor mice were injected into recipient mice through a tail vein. Bone marrow from C57B6 wild-type mice injected into C57B6 wild-type recipients gave M+/H+ chimeric mice. Bone marrow from C57B6 wild-type mice injected into ICAM-1 knock-out recipients gave M+/H– chimeric mice. Bone marrow from ICAM-1 knock-out mice injected into C57B6 wild-type recipients gave M–/H+ chimeric mice. All groups of chimeric mice were then treated with LPS or saline and 6 h later left ventricular contractility and function were measured and hearts were harvested as described above.

2.5. Cardiac ICAM-1 expression
To measure ICAM-1 protein expression, 10 µm thick frozen sections were prepared and fixed using 3% paraformaldehyde for 20 min and then incubated with Universal Blocking Agent (Dako, Carpinteria, CA) for 2–3 h. Sections were treated with rat anti-mouse ICAM-1 antibody (1/500) (BD Pharmingen) at 4 °C overnight followed by incubation with goat anti-rat fluorescently labeled antibody (1/1000) (Alexa-fluor 594, Molecular Probes, Eugene, OR) and Hoechst nuclear stain (1/1000) (H3570, Molecular Probes) for 3 to 4 h at room temperature. Images were captured at 400x using a confocal microscope (SP-2, Leica Corporation, Exton, PA). The ratio of mean intensity of ICAM-1 staining (red) to mean intensity of nuclear staining (blue) was measured (Fluocytogram, Leica Corporation, DIMRE2, Exton, PA).

2.6. Cardiac leukocyte infiltration
Sections (6 µm) were prepared from formalin fixed hearts and stained with hematoxylin and eosin. Images were captured at 400x using a spot camera and measurements were made using Image-Pro Plus software (Media Cybernetic Inc., Silver Spring, MD). The number of neutrophils within coronary arteries, the number of neutrophils adherent to the endothelial surface, and the number of neutrophils within the perivascular space (defined as an area of double the largest radius of the corresponding coronary artery) were measured.

2.7. Fibrinogen immuno-histochemistry
Formalin fixed hearts were embedded in paraffin and 6 µm sections were prepared. After de-parafinization, sections were treated with rabbit anti-mouse fibrinogen (Inovation Research Southfield, MI) overnight followed by HRP-conjugated anti-rabbit antibody (BD Pharmingen, San Jose, CA) for 2 h. The slides were then treated with DAB for 30 min followed by hematoxylin staining. Images (x400) were captured using a spot camera.

2.8. Statistical analysis
We tested for differences in Ees, [dP/dtmax]/EDV, and ejection fraction between groups using analysis of variance, choosing p<0.05 as significant. When a significant difference was found we identified specific differences between groups using a sequentially rejective Bonferroni test procedure. Data are expressed as mean±standard error throughout.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Endotoxin increases myocardial ICAM-1 expression in C57B6 wild-type mice but not ICAM-1 KO mice
3.1.1. C57B6 wild-type mice
Immunofluorescent staining of frozen sections from LPS-treated and saline-treated C57B6 wild-type mice demonstrated that ICAM-1 expression in heart sections significantly increased 6 h after LPS (Fig. 2). The ratio of the mean intensity of ICAM-1 staining to the mean intensity of nuclear staining was greater in the LPS-treated mice (4.59±0.32) than in control saline-treated mice (1.96±0.16, p<0.001). The intensity of ICAM-1 staining was heterogeneously distributed within each LPS-treated mouse heart section.


Figure 2
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  		Fig. 2 Representative immunofluorescent staining of myocardial sections from A. C57B6 saline-treated mice, B. C57B6 LPS-treated mice are shown in the top panel. The ratio of ICAM-1 to nuclear staining is shown for all mice in the above groups in the bottom panel. This ratio is significantly greater in C57B6 LPS-treated mice compared to saline-treated control mice (p<0.01). This ratio is greatly decreased in ICAM-1 knock-out mice and is not altered by LPS.

 
3.1.2. ICAM-1 KO mice
The ratio of ICAM-1 to nuclear immunofluorescent staining was greatly decreased in ICAM-1 knock-out mice (0.15±0.09) compared to C57B6 wild-type mice (1.96±0.16, p<0.05) and did not increase with LPS injection (0.18±0.13) (Fig. 2).

3.2. Endotoxin results in reduced cardiac contractility in C57B6 wild-type mice but not in ICAM-1 KO mice
3.2.1. C57B6 wild-type mice
We determined whether this endotoxemic murine model of sepsis resulted in decreased left ventricular contractility and function comparable to large animal models [16,17] and human sepsis [2,18]. Six hours after LPS injection in C57B6 wild-type mice, heart rate increased by 72±6% (p<0.01) and end-systolic pressure decreased by 12±4% compared to saline-treated controls (Table 1). Left ventricular contractility decreased after LPS injection as indicated by a 58±4% decrease in Ees (p<0.05; Fig. 3A) and a 60±6% decrease in [dP/dtmax]/EDV. Ejection fraction decreased by 14±10% despite the decrease in end-systolic pressure (Table 1) confirming the finding of a substantially reduced left ventricular systolic contractility.


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  		Table 1 Hemodynamics and ventricular function measures

 

Figure 3
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  		Fig. 3 Stylized pressure–volume loops drawn using average end-systolic elastance (Ees), end-diastolic volume (EDV), end-diastolic pressure (EDP) and end-systolic pressure (ESP) as reported in Table 1. End-systolic volume (ESV) derived from these plots corresponds closely (but not exactly due to averaging) to the measured ESV values reported in Table 1. A. In C57B6 wild-type mice, LPS results in a significant decrease in left ventricular contractility as measured by Ees. This is accompanied by an increase in ESV and EDV. B. In ICAM-1 knock-out mice, LPS does not result in a decrease in left ventricular contractility measured by Ees.

 
3.2.2. ICAM-1 KO mice
In contrast to C57B6 wild-type mice, left ventricular contractility and function in ICAM-1 knock-out mice, as measured by Ees (Fig. 3B), [dP/dtmax]/EDV 20% change p=NS, and ejection fraction (Table 1), were not decreased. Similarly, 6 h after LPS injection heart rate changed less in ICAM-1 knock-out mice (increased 14%) compared to C57B6 wild-type mice (increased 72%) while end-systolic pressure decreased to the same extent in ICAM-1 knock-out mice (by 24%) compared to C57B6 wild-type mice (by 23%).

3.3. Leukocytes mediate the endotoxin induced reduction in cardiac contractility
To test the hypothesis that leukocytes contribute to the early (6 h) decrease in left ventricular contractility in vivo in this intact whole animal model of sepsis, we increased leukocyte counts using GCSF injection or decreased leukocyte counts using cyclophosphamide injection. The GCSF injection increased total peripheral leukocyte count by 31±10% and increased peripheral neutrophil count by 20% over untreated control C57B6 wild-type mice (Table 2). The GCSF increased bone marrow leukocyte count 3.5 fold and increased bone marrow neutrophil count 4 fold. In GCSF-treated mice LPS injection resulted in an 59±15% reduction in Ees (p<0.05), a 21±19% decrease in [dP/dtmax]/EDV, and an 36±9% decrease in ejection fraction compared to controls (p<0.05; Table 1).


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  		Table 2 Peripheral blood cellular components

 
Cyclophosphamide treatment decreased the total leukocyte count by 97±1% to 0.08±0.01x109/L, and decreased the fraction of neutrophils to 34.7±0.9% (Table 2). Cyclophosphamide pretreatment abolished the effect of LPS, which did not decrease Ees, [dP/dtmax]/EDV, or ejection fraction (Table 1) in these mice.

3.4. Myocardial but not leukocyte expression of ICAM-1 mediates endotoxin induced cardiac dysfunction
To assess whether the ICAM-1 mediated cardiac dysfunction was predominantly leukocyte or tissue in origin we created ICAM-1 bone marrow chimeric animals. M+ indicates that marrow-derived cells can express ICAM-1; M– indicates that marrow-derived cells can not express ICAM-1; H+ indicates the heart cells can express ICAM-1 while H– indicates that heart cells do not express ICAM-1. LPS injection decreased left ventricular contractility of M+/H+ and M–/H+ chimeric mice. Specifically, Ees decreased by 43±7% (p<0.05) in M+/H+ chimeric mice and Ees decreased by 56±13% (p<0.05) in M–/H+ chimeric mice 6 h after LPS injection. These changes in left ventricular contractility in M+/H+ and M–/H+ mice were not different from the effect of LPS injection in C57B6 wild-type mice (Table 1). In contrast, LPS injection did not decrease left ventricular contractility of M+/H– chimeric mice. Indeed, in M+/H– chimeric mice LPS injection resulted in increases in contractility as measured by Ees (24.0±3.7 in LPS group versus 13.0±2.2 in controls), [dP/dtmax]/EDV (1010±180 in LPS group versus 500±90 in controls) and ejection fraction (54±8% in LPS group versus 45±4% in controls, p=NS, Table 3). Taken together, the lack of LPS-induced myocardial dysfunction in H– mice compared to the significant LPS-induced myocardial dysfunction in both M+ or M–/H+ mice indicates that cardiac ICAM-1 expression is an important factor leading to decreased left ventricular contractility following LPS injection.


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  		Table 3 Hemodynamics and ventricular function measures in chimeric mice

 
Injection of the bone marrow concentrate into the chimeric recipient mice significantly increased the peripheral blood total leukocyte count compared to non-injected cyclophosphamide-treated mice, confirming successful bone marrow transplantation. In M+/H– mice the peripheral blood total leukocyte count was 1.2±0.04x109/L, the fraction of neutrophils was 29.8±3.7%, and the fraction of lymphocytes was 23.3±3.6%. In M+/H+ mice the peripheral blood total leukocyte count was 1.2±0.05x109/L, the fraction of neutrophils was 46.8±5.6%, and the fraction of lymphocytes was 18.9±5.3% (Table 4).


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  		Table 4 Peripheral blood cellular components in chimeric mice

 
3.5. ICAM-1 mediated cardiac dysfunction does not require neutrophil extravasation, but may be mediated through increased interstitial fibrinogen
To determine whether the leukocyte mediated cardiac dysfunction was due to intramyocardial we used morphometric measures. LPS injection in C57B6 wild-type mice increased coronary intravascular neutrophil concentration from 2.5±1.9x10–5/µm2 in control to 137±22x10–5/µm2 in septic mice. LPS injection also increases the ratio of adherent neutrophil to endothelial surface from 3.2±2.3x10–5/µm2 in control to 99±18x10–5/µm2 in endotoxemic mice (p<0.05). However, LPS injection did not increase extracellular neutrophil infiltration into the cardiac tissue (Fig. 4). As fibrinogen is an acute phase protein known to bind ICAM-1, we looked for evidence of increased fibrinogen in the myocardial interstitium where it can directly interact with myocyte surface ICAM-1. While saline-treated C57B6 animals showed virtually no fibrinogen there was a dramatic increase in cardiac interstitial fibrinogen in LPS-treated animals (Fig. 5).


Figure 4
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  		Fig. 4 LPS injection significantly increased the number of neutrophils within the intravascular space (p<0.01) and the number of neutrophils adherent to vascular endothelium (p<0.01). This effect is eliminated in ICAM-1 knock-out mice. The number of extravascular neutrophils was low in all groups. p<0.01 LPS versus saline.

 

Figure 5
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  		Fig. 5 Representative immunohistochemical staining for fibrinogen in cardiac sections from C57BL6 mice. A) 6 h post LPS treatment, B) 6 h following saline injection. Fibrinogen stains brown on a blue hematoxylin counterstain, and is dramatically increased in the cardiac interstitium of LPS-treated mice.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
LPS injection reduced left ventricular contractility in this whole animal model of systemic inflammation, similar to the previous measurements in a variety of animal models of sepsis, and similar to the decrease in left ventricular function observed during human septic shock. Decreasing leukocyte count using cyclophosphamide abrogated the effect of LPS, confirming previous reports that leukocytes contribute to the development of decreased left ventricular contractility in sepsis. ICAM-1 knock-out mice were resistant to the effect of LPS on left ventricular contractility indicating that ICAM-1 plays an important causal role in mediating the effect of LPS. Chimeric mouse constructs further implicate cardiac ICAM-1 expression, versus ICAM-1 expressed on bone marrow-derived cells transiting the heart. Thus, signaling via cardiac ICAM-1 is necessary for the development of leukocyte-dependent decreases of left ventricular contractility in this whole animal model of systemic inflammation.

Many mechanisms have been proposed to contribute to myocardial depression during sepsis [19]. In sepsis, release of pathogenic toxins such as LPS from gram negative bacteria, glycolipid from gram positive bacteria, or mannan from fungal wall initiates a systemic inflammatory response. Circulating pro-inflammatory cytokines trigger NOS III mediated NO production within minutes [20], and NOS II sustains NO levels over the following hours [21]. NO is an important mediator of myocardial dysfunction in sepsis [21–23]. Leukocyte derived reactive oxygen intermediates contribute directly and via formation of peroxynitrite radicals, to myocardial damage and dysfunction [10,23]. Coronary capillary endothelial activation, damage and dysfunction also contribute, in part by impaired regulation of coronary microvascular blood flow, which impairs myocardial oxygen extraction [17].

Local accumulation of leukocytes has been proposed to impair myocyte function through various means. Leukocyte retention within coronary capillaries is increased upon endotoxin infusion [16], and is associated with myocardial morphometric damage and decreased ventricular contractility [5]. Ex vivo, filtering leukocytes from coronary blood perfusing the heart prevented the decrease in contractility within the first 6 h of endotoxin infusion [6]. Activated neutrophils appear to be a particularly important leukocyte subset involved in early myocardial depression [4]. Our current results support these previous observations by demonstrating that the LPS-induced decrease in left ventricular contractility is leukocyte dependent – being abrogated by a decreased peripheral blood leukocyte count.

One proposed mechanism is local oxidant injury mediated via leukocyte-derived reactive oxygen species [7,8,10,24]. However, this effect is observed in vitro when one or more leukocytes adhere to each cardiomyocyte either leukocyte-expressed {alpha}4 integrins binding to fibronectin found on cardiac myocytes, or leukocyte expressed integrins binding to ICAM-1 on cardiac myocytes.[7,8,10,24]. We found very few intramyocardial leukocytes, suggesting that oxidant injury alone as the primary cause of the in vivo myocardial suppression observed in this study is unlikely. Alternatively, part of the leukocyte effect may be due to binding of leukocytes to cardiac ICAM-1. Pro-inflammatory cytokines, including IL-6 generated by the myocardium following ischemia–reperfusion, result in CD18-ICAM-1 dependent adhesion of PMN to cardiomyocytes [25]. Co-culture of macrophages or neutrophils with cardiomyocytes resulted in ICAM-1 dependent adhesion and decreased cardiomyocyte fractional shortening [7,9]. Adhesion alone was a fundamentally important aspect of this interaction because the same effect was reproduced by co-culture with killed and fixed neutrophils or simply by ICAM-1 cross-linking antibodies [9].

ICAM-1 is a 76–110 kDa glycoprotein from the immunoglobulin gene family [26,27]. ICAM-1 expression by a variety of different cell types, including on cardiomyocytes, increases during inflammation [7–9]. Activated coronary endothelial cells in cell culture increase ICAM-1 expression in 2 to 4 h and maximum ICAM-1 expression is reached in 6 to 8 h. High levels of ICAM-1 expression are maintained as long as the inflammatory response is active [28]. Activation of ICAM-1 stimulates further production and expression of vascular cellular adhesion molecule-1 (VCAM-1) and ICAM-1 [29]. Activation of isolated cardiomyocytes increases ICAM-1 mRNA production within 1 to 4 h [9] and expression of ICAM-1 protein within 4–6 h [7]. Maximum production of ICAM-1 occurs 18 to 24 h after cytokine stimulation [8]. Ligands for ICAM-1 include CD11/CD18 receptors [26,29,30], fibrinogen [31], rhinovirus receptors [32] and plasmodium falciparum [33].

Our current findings extend these in vitro observations and demonstrate that binding of ICAM-1 expressed on cardiomyocytes reduced left ventricular contractility in a whole animal model of systemic inflammation. Using isolated rat cardiomyocytes we have shown that activation of ICAM-1 using cross-linking antibodies can initiate intracellular signals which cause discoordinate calcium influx leading to reduction in cardiomyocyte contractility [9]. This effect of ICAM-1 cross-linking is mediated via the cytoskeleton [9]. The presence of cardiac contractile dysfunction after LPS injection in M+/H+, and M–/H+ chimera mice, and absence in M+/H– chimera mice demonstrate the importance of ICAM-1 expression within cardiac tissue. Lack of ICAM-1 on bone marrow-derived inflammatory cells did not prevent the effect of LPS on cardiac contractility.

Several potential ligands may be involved in ICAM-1 binding and activation in this whole animal model of systemic inflammation, which results in decreased left ventricular contractility. Since we [6] and others [5] have demonstrated that the LPS-induced decrease in left ventricular contractility is, in part, leukocyte dependent, it is reasonable to postulate that CD11/CD18, or other leukocyte expressed ICAM-1 ligands, bind and activate ICAM-1 expressed on cardiomyocytes in vivo. However, we did not find many neutrophils or other leukocytes within the cardiac parenchyma in this and other models of sepsis [4]. It is conceivable that leukocytes enter cardiac parenchyma but undergo apoptosis or other forms of cell death quickly and leave CD11/CD18 to bind to ICAM-1 expressed on cardiomyocytes. However, this has not been demonstrated. We propose an alternative mechanistic pathway to account for the observation of leukocyte-dependent decreases in left ventricular contractility mediated by cardiomyocyte expressed ICAM-1 binding and activation. Current and previous observations demonstrate that leukocytes are retained within coronary capillaries in systemic inflammatory states [4]. These leukocytes damage the capillary endothelium [4] leading to interstitial edema. We postulate that the interstitial edema fluid components that are ICAM-1 ligands, including fibrinogen, could then lead to decreased ventricular contractility via the ICAM-1 binding. This postulated pathway is consistent with the observations of Neviere and his colleagues [5].

In summary, we found that LPS injection in mice resulted in a substantial decrease in left ventricular contractility. This decrease was leukocyte dependent and also dependent on cardiac ICAM-1 expression. However, we did not observe a large number of leukocytes within the cardiac parenchyma at the time of substantially decreased left ventricular contractility. Therefore, we postulate that this leukocyte-dependent decrease in left ventricular contractility involves at least two steps in series – a leukocyte dependent step and an ICAM-1 dependent step. Leukocyte retention leading to endothelial damage and leakage of ICAM-1 ligands, such as fibrinogen, into the cardiac interstitium is one explanation compatible with our findings. We conclude that signaling via cardiac ICAM-1 expression is necessary to mediate leukocyte-dependent decreases of left ventricular contractility in endotoxemic mice.


   Notes
 
* The support from Canadian Institutes of Health Research is acknowledged. K.R. Walley is a Michael Smith Foundation for Health Research distinguished scholar.

Time for primary review 21 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

  1. Angus D.C., Wax R.S. Epidemiology of sepsis: an update. Crit Care Med (2001) 29:S109–S116.[CrossRef][ISI][Medline]
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