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Am J Physiol Heart Circ Physiol 287: H1013-H1022, 2004. First published April 15, 2004; doi:10.1152/ajpheart.01177.2003
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Novel regulatory mechanism of cardiomyocyte contractility involving ICAM-1 and the cytoskeleton

Ehsan Y. Davani, Delbert R. Dorscheid, Cheng-Han Lee, Cornelis van Breemen, and Keith R. Walley

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

Submitted 16 December 2003 ; accepted in final form 8 April 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
ICAM-1 mediates interaction of cardiomyocytes with the extracellular matrix and leukocytes and may play a role in altering contractility. To investigate this possibility, rat ventricular cardiomyocytes were activated using TNF-{alpha}, IL-1{beta}, or LPS, washed, cultured with quiescent rat polymorphonuclear leukocytes (PMNs) for 4 h, and electrically stimulated to determine fractional shortening. PMNs cultured with activated cardiomyocytes reduced control fractional shortening of 20.5 ± 0.7% by –2.8 ± 0.3% per adherent PMN (P < 0.001). Fixing PMNs with paraformaldehyde or glutaraldehyde did not prevent PMN-mediated decreases in cardiomyocyte fractional shortening. However, PMN adherence and decreased fractional shortening were prevented by anti-ICAM-1 and anti-CD18 antibodies. Reduced fractional shortening was reproduced in the absence of PMNs by ICAM-1 binding using cross-linking antibodies (reduced by 36 ± 3% from control, P < 0.01). Immunofluorescent staining demonstrated increased cortical cytoskeleton-associated focal adhesion kinase expression after ICAM-1 cross-linking, suggesting involvement of the actin cytoskeleton. Indeed, disruption of F-actin filament assembly using cytochalasin D or latrunculin A did not prevent PMN adherence but prevented decreased fractional shortening. Inhibition of the cytoskeleton-associated Rho-kinase pathway with HA-1077 prevented ICAM-1-mediated decreases in cardiomyocyte contractility, further suggesting a central role of the actin cytoskeleton. Importantly, ICAM-1 cross-linking did not alter the total intracellular Ca2+ transient during cardiomyocyte contraction but greatly increased heterogeneity of intracellular Ca2+ release. Thus we have identified a novel regulatory mechanism of cardiomyocyte contractility involving the actin cytoskeleton as a central regulator of the normally highly coordinated pattern of sarcoplasmic Ca2+ release. Cardiomyocyte ICAM-1 binding, by PMNs or other ligands, induces decreased cardiomyocyte contractility via this pathway.

intracellular signaling; focal adhesion kinase; Rho; calcium

INTRAMYOCARDIAL INFLAMMATION, for example, after ischemia-reperfusion, in inflammatory cardiomyopathy, during orthotopic heart transplant rejection, and during sepsis-induced myocardial dysfunction, results in increased expression of ICAM-1 on cardiomyocytes (15, 22, 32, 42) and accumulation of intramyocardial leukocytes (18–21). Adhesion of polymorphonuclear leukocytes (PMNs) or macrophages to cardiomyocytes is mediated in part by binding to ICAM-1 on cardiomyocytes. If PMNs are highly activated, they kill cardiomyocytes within minutes by release of reactive oxygen intermediates (13, 16, 41). However, ICAM-1-mediated adhesion of relatively quiescent macrophage/monocytes to cardiomyocytes reduces contractility without killing the cardiomyocyte (47), and a recent report implicates ICAM-1 in mediating reduced cardiomyocyte contractility independent of PMN accumulation (42).

ICAM-1 is fundamentally important in linking extracellular mechanical signals to regulation of intracellular processes (26). ICAM-1 is closely associated with the actin cytoskeleton (2, 37), and ICAM-1 binding increases linkage between the cell surface ICAM-1 receptor and the cytoskeleton (1, 50). Whether the cytoskeleton plays a role in modulating cardiomyocyte contractility is unknown, and potential signaling pathways and mechanisms involved have not been identified. Accordingly, we postulated that ICAM-1 and the cardiomyocyte cytoskeleton may be involved in a novel pathway leading to cardiomyocyte contractile dysfunction. Although many mediators (e.g., cytokines, leukocytes, reactive oxygen intermediates, and nitric oxide) (7, 9, 36) and pathways (e.g., apoptosis and adrenergic signaling) (31, 35, 49) are involved in myocardial dysfunction during intramyocardial inflammation, we focused on the potential role of ICAM-1 binding and its relation to the cardiomyocyte cytoskeleton.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
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This study was approved by the University of British Columbia Animal Care Committee and adheres to Canadian and National Institutes of Health guidelines for animal experimentation.

Isolation of rat ventricular myocytes. Male Sprague-Dawley rats (250–300 g) were anesthetized using 3% halothane, and the heart was excised and mounted on a modified Langendorff apparatus and perfused with oxygenated (95% O2-5% CO2) HEPES-Joklik-modified MEM buffer (GIBCO-BRL, Grand Island, NY) at 37°C for 2 min. The perfusate was changed to 30 ml of recirculating Ca2+-free MEM containing 236 U/ml collagenase (Worthington Biochemical, Freehold, NJ). At 15 and 20 min, the Ca2+ concentration was increased stepwise to 0.025 and 0.075 mmol/l. After 30–40 min, the ventricles were removed from the perfusion system, gently teased apart, and agitated for 2–3 min at 37°C. The tissue and dispersed cells in solution were filtered through a 200-µm nylon mesh. The cells were washed three times at 37°C in MEM containing increasing Ca2+ concentrations (200 µM, 500 µM, and 1 mM), with the cells allowed to settle by gravity for 10 min between each wash. Cells were resuspended in 37°C HEPES-modified M199 buffer (GIBCO-BRL) with 1% BSA. The cells were diluted to a final concentration of 5 x 104 cells/ml, 100 µl were loaded into each well of 96-well plates, and the plates were incubated at 37°C in 95% O2-5% CO2. At 90 min, the medium was changed to fresh M199 with BSA, and the cardiomyocytes were incubated for 24 h to allow them to become relatively quiescent. After 24 h, the cells were considered viable if they demonstrated a characteristic rod shape without cytoplasmic blebbing. This morphometric assessment of viability was confirmed in a subset of experiments with trypan blue exclusion. We have found that the fraction of viable cardiomyocytes is always >85%.

Cardiomyocyte ICAM-1 protein expression. We and others have shown that activation of cardiomyocytes by inflammatory cytokines increases cardiomyocyte expression of ICAM-1 (42, 47, 53). To confirm this, we measured ICAM-1 protein expression on cardiomyocytes that were incubated with 20 ng/ml TNF-{alpha} for 4 h and then fixed using 3% paraformaldehyde for 30 min. Cardiomyocytes were incubated with 10 µg/ml mouse anti-rat ICAM-1 antibody (1A29 [PDB] , BD Pharmingen, San Jose, CA) for 3 h, washed, and incubated with a fluorescent secondary anti-mouse antibody (1:500; Alexa Fluor 488, Molecular Probes, Eugene, OR) for 3 h and imaged using confocal microscopy (DIMRE2, Leica, Exton, PA). The mean of intensity per pixel of each cardiomyocyte was measured using Image Pro-Plus (11).

Cardiomyocyte ICAM-1 mRNA expression. Cardiomyocytes (5 x 105) were cultured in 60-ml laminin-coated dishes and incubated with or without TNF-{alpha}. Cells were then lysed and frozen at –70°C. RNA was extracted using the standard technique of a Qiagen RNA kit (Qiagen, Mississauga, Ontario, Canada): 1 µl of eluted RNA was added to 999 µl of 0.1 M NaOH solution in diethyl pyrocarbonate-water; 3 µg of RNA were taken as constant template for the RT cycle to make cDNA; 2 µl of cDNA were made from total RNA and used as a template to amplify rat ICAM-1 and rat GAPDH using the following primer sequence: 5'-AGG TCA GGG GTG TCG AGC-3' (forward ICAM-1 primer) and 5'-CAA GGA GAT CAC ATT CAC GG-3' (reverse ICAM-1 primer). The PCR conditions for Perkin-Elmer model 9700 were as follows: 95°C for 15 min (Qiagen Taq polymerase enzyme activation), denaturing at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min. The whole cycle was repeated 40 times. PCR products were separated on 3% agarose gel stained with ethidium bromide and visualized using an Eagle Eye UC scanner. The ICAM-1 signal was normalized to the GAPDH signal for each time point sample. The final results of TNF-{alpha}-treated cardiomyocytes were compared with controls.

Measurement of cardiomyocyte fractional shortening. At 15 min before measurement of fractional shortening, 2 µl of 0.5% trypsin were added to each well. Preliminary experiments demonstrated that this concentration of trypsin did not alter cell viability and cleaved >95% of the adherent cardiomyocytes from their attachments to the bottom of the well. Specially designed platinum electrodes were then lowered into each well in the 96-well plate, and the cardiomyocytes were electrically stimulated (model S48 stimulator, Grass, West Warwick, RI; 45 V, 2.2-ms duration, 25-{Omega} resistance) while being recorded by video microscopy (model SLV-760HF, Sony) at x400 magnification. This electrical stimulus was chosen from preliminary threshold experiments as two times the minimum electrical stimulus required to maximally contract the cardiomyocytes. Still frames of systolic and diastolic cardiomyocytes were captured using Image PC (Scion, Frederick, MD), and cardiomyocyte fractional shortening was calculated as the difference between diastolic and systolic length divided by diastolic length.

Isolation of peripheral blood PMNs. Male Sprague-Dawley rats (250–300 g) were anesthetized as described above, and, via cardiac puncture, 8 ml of blood were drawn into a syringe containing 2 ml of anticoagulant citrate-dextrose solution (Baxter, Deerfield, IL). Leukocyte-rich plasma was obtained from the blood using dextran sedimentation. Red blood cells were lysed using sterile water, and PMNs were purified using Histopaque 1077 (Sigma-Aldrich, Oakville, ON, Canada) to remove the remaining erythrocytes and mononuclear cells. Purified PMNs were resuspended in medium 199 (M199) and used immediately. In further experiments, PMNs were fixed by incubation in 3% paraformaldehyde for 15 min or by incubation in 0.025% glutaraldehyde for 30 min.

CD11b expression on PMNs. To confirm that this very low concentration of glutaraldehyde preserves the extracellular domains of PMN CD11b (23), freshly isolated PMNs and glutaraldehyde-fixed PMNs were incubated with mouse anti-rat CD11b R-phycoerythrin-conjugated antibody (Biosource, Camarillo, CA) or nonspecific mouse antibody (IgG negative control; Biosource) at 30 µl/ml for 30 min. Cells were washed with PBS, fixed with 3% paraformaldehyde, and read using a flow cytometer (EPICS XL-MCL, Beckman Coulter, Miami, FL).

Coculture of PMNs and cardiomyocytes. After 24 h of cardiomyocyte incubation, 5 x 104 freshly isolated PMNs were added to each well of cardiomyocytes (10 PMNs per cardiomyocyte) in the 96-well laminin-coated plates for coculture experiments. During offline analysis of captured video sequences, we viewed all cardiomyocytes throughout a contraction. To be counted as adherent, PMNs had to move with the contracting cardiomyocyte and maintain a contact-relative location on the cardiomyocyte membrane during contraction.

ICAM-1 cross-linking. Following previously reported methods of cross-linking ICAM-1 and initiating intracellular signaling (17, 30, 51), we incubated cardiomyocytes with 1 µg/ml mouse anti-rat ICAM-1 or 1 µg/ml control mouse nonspecific IgG (Dako, Carpinteria, CA) for 1 h. Cells were washed and then incubated with 10 µg/ml goat anti-mouse IgG (Transduction Laboratories, San Jose, CA) for 4 h to cross-link the bound anti-ICAM-1.

Immunofluorescent imaging of focal adhesion kinase. Cardiomyocytes were cultured on eight-well laminin-coated slides (Lab-Tek, Naperville, IL). After 24 h, ICAM-1 on cardiomyocytes was cross-linked as described above. Cells were fixed and permeabilized with 3% paraformaldehyde for 20 min and 0.5% Triton X-100 for 5 min in microtubule-stabilizing buffer (0.1 M MES: 2 mM EGTA, 2 mM MgCl2, and 4% polyethylene glycol 8000; Sigma-Aldrich). After the cells were blocked with PBS + 0.5% BSA for 60 min, 40 µg/ml rabbit anti-focal adhesion kinase (FAK; UBI, Lake Placid, NY) was added and incubated overnight at 4°C. After the cells were washed, Alexa Fluor 488-labeled secondary goat anti-rabbit antibody (10 µg/ml; Molecular Probes) was added for 45 min at room temperature.

Images were captured using a Noran Oz laser scanning confocal microscope with a high-power objective lens (Nikon, Plan Fluor, x100 oil, numerical aperture 1.30) and slit width of 10 µm. For visualization of the Alexa Fluor 488-labeled specimens, the sample was illuminated using 488-nm light from an argon-krypton laser. A high-gain photomultiplier tube collected the emission after it had passed through a 525/52-nm band-pass filter. All parameters (e.g., laser intensity and gain) were left unchanged during the experiment and were set so that the level of photobleaching was negligible.

To obtain high-resolution images within the cortical cytoplasm, multiple images were acquired along the z-axis at 0.5-µm steps. z-Stack images were deconvolved using XCOSM (10), and a reconstructed image 2 µm from the top surface of each cardiomyocyte was evaluated. Fluorescence intensity of FAK staining was then measured as described above.

Cardiomyocyte Ca2+ transient. Cardiomyocytes (1 x 104) were cultured in each well of eight-well laminin-coated slides. After ICAM-1 cross-linking or control, cardiomyocytes were incubated with 2 µM fluo 4 (F14201 [GenBank] , lot 28A2–5, Molecular Probes) for 10 min, washed three times, and electrically stimulated as described above. One hundred fifty time series images were captured at 33-ms intervals during cardiomyocyte contraction using laser scanning confocal microscopy. To quantitate coordination of simultaneity of intracellular Ca2+ release, three regions of interest were selected at the center and at each end of each cardiomyocyte. The mean intensity per pixel in each region of interest was measured from the beginning of contraction to the end of relaxation in each cardiomyocyte. The relative dispersion between the three regions of interest was used to quantitate heterogeneity. The time integral of the Ca2+ intensity curve was used to quantitate total Ca2+ release.

Data analysis. We tested for differences in fractional shortening and number of adherent PMNs between control and treated groups of cardiomyocytes using ANOVA; we chose P < 0.05 as significant. When a significant difference was found, we identified specific differences between groups using a sequentially rejective Bonferroni test procedure. Values are means ± SE.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Uniform coculture conditions. We conducted preliminary experiments to establish uniform coculture conditions for the experiments in this study. Preliminary experiments demonstrated that, after 24 h of incubation, cardiomyocyte fractional shortening was 20.5 ± 0.7% (n = 36). In the absence of PMNs, fractional shortening did not significantly change after activation of quiescent cardiomyocytes by 2 h of incubation with 20 ng/ml TNF-{alpha} (BD Pharmingen; 91.3 ± 4% of control, P = NS), 20 ng/ml IL-1{beta} (Sigma-Aldrich; 91.1 ± 6% of control, P = NS), or 10 µg/ml LPS (Sigma-Aldrich; 86.5 ± 5% of control, P = NS). At 1 h after TNF-{alpha} activation, the ratio of cardiomyocyte ICAM-1 to GAPDH mRNA increased from 0.091 ± 0.08 to 0.361 ± 0.10. Similarly, after TNF-{alpha} activation, ICAM-1 protein expression significantly increased (Fig. 1).



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  		Fig. 1. Top: ICAM-1 protein expression on cardiomyocytes increases 4 h after activation with TNF-{alpha} (B) compared with nonactivated controls (A). Bottom: significant increase in ICAM-1 protein expression after TNF-{alpha} activation.

 
When nonactivated cardiomyocytes were cultured with freshly isolated PMNs for 4 h, fractional shortening did not change (20.7 ± 1.2%, n = 36; Fig. 2A). However, TNF-{alpha}-activated cardiomyocytes in culture with freshly isolated PMNs did decrease fractional shortening. On the basis of the percentage of cardiomyocytes with normal morphology, the number of PMNs adherent to each cardiomyocyte, and maximal contractile dysfunction (Fig. 2), all subsequent experiments were conducted after a 4-h incubation of TNF-{alpha}-activated cardiomyocytes with freshly isolated PMNs.



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  		Fig. 2. Results from preliminary experiments to determine conditions for all further experiments. A: polymorphonuclear leukocytes (PMNs) in coculture with quiescent cardiomyocytes ({blacktriangleup}) did not alter fractional shortening, whereas fractional shortening was significantly reduced in PMNs in coculture with activated cardiomyocytes (which induces cardiomyocyte ICAM-1 expression) for 4 h ({blacksquare}). B: number of PMNs adherent to activated cardiomyocytes increased to a maximum at 4 h, and cardiomyocyte viability remained high until 4 h in coculture. On the basis of these results, further experimental measurements used PMNs in coculture with TNF-{alpha}-activated cardiomyocytes for 4 h.

 
Adherent PMNs reduce cardiomyocyte fractional shortening. When activated cardiomyocytes were cultured with freshly isolated PMNs, fractional shortening decreased significantly in cardiomyocytes treated with TNF-{alpha} (–34.2 ± 5%, P < 0.001), IL-1{beta} (–48.6 ± 3.2%, P < 0.001), and LPS (–23.1 ± 3.9%, P < 0.001). Average PMN adhesion increased from 1.1 ± 0.2 PMNs per cardiomyocyte in the nonactivated cardiomyocytes to 2.7 ± 0.2, 1.4 ± 0.1, and 1.9 ± 0.2 in cardiomyocytes treated with TNF-{alpha}, IL-1{beta}, and LPS, respectively (P < 0.05 for each). Fractional shortening of cardiomyocytes in culture with PMNs decreased by –2.8 ± 0.3% per adherent PMN (P < 0.001; Fig. 3A).



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  		Fig. 3. A: fractional shortening of cardiomyocytes decreased as the number of attached PMNs per cardiomyocyte increased. B: the same effect was present even when PMNs were first killed and fixed using glutaraldehyde or paraformaldehyde. Results implicate PMN-cardiomyocyte binding as a key step in mediating decreased cardiomyocyte fractional shortening. Numbers within bars indicate numbers of cardiomyocytes.

 
Adherent PMNs may have contributed to decreased cardiomyocyte contractility simply by release of reactive oxygen intermediates or other mediators in close proximity to the cardiomyocyte (16). Alternatively, adhesion itself may have contributed. To help distinguish between these two possibilities, we fixed PMNs before coculture. First, PMNs were fixed with 3% paraformaldehyde for 15 min. After they were washed five times, fixed PMNs were cultured with cardiomyocytes. Paraformaldehyde-fixed PMNs decreased fractional shortening by 27 ± 2% compared with control (P < 0.05), which is comparable to the effect of freshly isolated PMNs. Glutaraldehyde-fixed PMNs similarly decreased cardiomyocyte fractional shortening by 25 ± 4% (P < 0.05) compared with control. The relation between the number of adherent PMNs and the decrease in fractional shortening was preserved (Fig. 3B), suggesting that this effect was due to adhesion of PMNs to cardiomyocytes. To exclude the possibility that glutaraldehyde fixation may have prevented binding by altering the extracellular domain of CD11b on PMNs, we used a low concentration of glutaraldehyde and used flow cytometry to confirm unchanged CD11b expression on PMNs (Fig. 4).



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  		Fig. 4. Flow cytometry of freshly isolated (A and C) and glutaraldehyde-fixed (B and D) PMNs. A: freshly isolated PMNs (identified from sidescatter-backscatter plots) incubated with nonspecific IgG. C: freshly isolated PMNs incubated with antibody to CD11b; note increased fluorescent intensity specific for CD11b. B: glutaraldehyde-fixed PMNs incubated with nonspecific IgG. D: same increase in fluorescent intensity specific for CD11b seen with freshly isolated PMN (C). PE, phycoerythrin.

 
ICAM-1 binding mediates decreased cardiomyocyte contractility. Antibody to ICAM-1 and antibody to CD-18 (BD Pharmingen), to block this important PMN-expressed ligand of ICAM-1, prevented the PMN-induced decrease in cardiomyocyte fractional shortening (Fig. 5A). The average number of PMNs adherent to each TNF-{alpha}-activated cardiomyocyte was also reduced by antibody to ICAM-1 and antibody to CD-18: 2.7 ± 0.2, 1.2 ± 1.0, and 1.1 ± 1.0 adherent PMNs per cardiomyocyte for control, antibody to ICAM-1, and antibody to CD-18, respectively. Thus the contractile effect of PMN binding involves binding to cardiomyocyte ICAM-1.



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  		Fig. 5. A: decrease in cardiomyocyte fractional shortening in TNF-{alpha}-activated cardiomyocyte-PMN coculture group (*P < 0.05) was prevented by antibody to ICAM-1 or antibody to its important ligand CD18. B: even in the absence of PMNs, ICAM-1 cross-linking reduces cardiomyocyte fractional shortening (*P < 0.05). This effect is prevented by disruption of the actin cytoskeleton by cytochalasin D or latrunculin A.

 
To demonstrate that PMN binding and the decrease in contractility in culture with glutaraldehyde-fixed PMNs were still ICAM-1 dependent, we repeated these experiments with and without antibody to ICAM-1. The number of adherent glutaraldehyde-fixed PMNs was reduced from 1.8 ± 0.2 to 0.58 ± 0.1 by antibody to ICAM-1, and the decrease in fractional shortening was prevented: 16.8 ± 1.1% in the presence of PMN without anti-ICAM-1 and 22.0 ± 1.1% with anti-ICAM-1 (P < 0.05).

To determine whether ICAM-1 binding itself can cause decreased contractility, we used cross-linking antibodies to ICAM-1 in the absence of PMNs. ICAM-1 cross-linking decreased fractional shortening by 36 ± 3% compared with control groups at 4 h (Fig. 5B). In contrast, replacement of anti-ICAM-1 antibodies with nonspecific IgG followed by addition of cross-linking antibodies did not change fractional shortening compared with control.

ICAM-1 cross-linking alters the cortical cytoskeleton. ICAM-1 is connected to cytoskeletal proteins (2, 6, 37, 50). To test for involvement of the cortical cytoskeleton after ICAM-1 cross-linking, we examined the distribution of the actin cytoskeleton-associated protein FAK within cardiomyocytes. Immunofluorescent imaging of FAK at 2 µm below the cardiomyocyte surface demonstrated increased cytoskeleton-associated FAK staining in ICAM-1 cross-linked cardiomyocytes compared with controls (Fig. 6).



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  		Fig. 6. Top: focal adhesion kinase (FAK) staining in control (left) and ICAM-1 cross-linked (right) cardiomyocytes. Bottom: average results for all cardiomyocytes (n = 64 for each group from 6 experiments) indicating that ICAM-1 cross-linking increases FAK expression associated with the cortical actin cytoskeleton 2 µm below cardiomyocyte surface. *P < 0.05.

 
Functional role of the cardiomyocyte actin cytoskeleton. To test the hypothesis that the actin cytoskeleton is involved in signaling pathways that lead to decreased cardiomyocyte contractility induced by ICAM-1 binding, we preincubated cardiomyocytes with 10 µM cytochalasin D, an actin-depolymerizing agent, for 2 h. Control cardiomyocyte fractional shortening was not altered by this concentration of cytochalasin D (106 ± 10% of control fractional shortening, n = 39 vs. n = 60 controls), confirming previous observations that these concentrations of cytochalasin D did not have a measurable effect on the function of sarcomeric F-actin (5). Cytochalasin D prevented the decrease in cardiomyocyte contractility due to ICAM-1 cross-linking (Fig. 5B). We repeated these experiments, disrupting the actin cytoskeleton in an independent way using 10 µM latrunculin A (Calbiochem, San Diego, CA), an agent that sequesters monomeric actin and inhibits elongation of actin filaments (52). Latrunculin A by itself did not significantly alter cardiomyocyte fractional shortening (111 ± 10% of control fractional shortening, n = 29 vs. n = 22 controls). Again, latrunculin A prevented the decrease in cardiomyocyte contractility due to ICAM-1 cross-linking (Fig. 5B).

Similarly, in experiments of PMNs cultured with activated cardiomyocytes, cytochalasin D and latrunculin A prevented the PMN-induced decrease in cardiomyocyte fractional shortening (20.5 ± 0.6, 13.6 ± 0.8, 17.5 ± 1.2, and 19.9 ± 2.7% in control, cardiomyocyte-PMN coculture, cytochalasin D, and latrunculin A, respectively) but did not alter the number of PMNs adherent to cardiomyocytes (1.6 ± 0.4, 1.8 ± 0.5, and 1.3 ± 0.8 in control, cytochalasin D, and latrunculin A, respectively).

Possible downstream signaling pathways. Rho family (small G) proteins are associated with the actin cytoskeleton and regulate cytoskeletal reorganization and gene expression. To investigate the role of these pathways, we used fusadil (HA-1077, Sigma-Aldrich), an inhibitor of Rho-kinase, in ICAM-1 cross-linked cardiomyocytes. HA-1077 (50 µM) completely prevented ICAM-1 binding-induced decreases in cardiomyocyte fractional shortening (Fig. 7), suggesting that this actin cytoskeleton-associated signaling pathway mediates ICAM-1 binding-induced decreases in cardiomyocyte fractional shortening.



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  		Fig. 7. Effect of ICAM-1 cross-linking on fractional shortening is prevented by RhoA inhibitor (HA-1077) but not by MAPK inhibitor (PD-98059). *P < 0.05 compared with control.

 
FAK activation leads to activation of a series of proteins and pathways, including MAPK pathways in other cell lines (8, 46). To investigate the role of these pathways in contractility of ICAM-1 cross-linked cardiomyocytes, we treated ICAM-1 cross-linked cardiomyocytes with the ERK1/2 inhibitor PD-98059 (10 µM; New England BioLabs, Mississauga, Ontario, Canada). ERK1/2 inhibition had no effect on the ICAM-1-induced decreases in cardiomyocyte fractional shortening (Fig. 7), suggesting that this signaling pathway was not directly involved.

ICAM-1 activation changes the pattern of Ca2+ release. To better understand how ICAM-1 binding could alter contractility, we measured the cardiomyocyte intracellular Ca2+ transient. ICAM-1 cross-linking changed the pattern of Ca2+ release in contracting cardiomyocytes. Ca2+ release changed from homogeneous and synchronized in control cardiomyocytes to a focal starting point propagating wave in ICAM-1 cross-linked cardiomyocytes (Fig. 8). The focal starting point propagating wave occurred in 86 ± 3% of ICAM-1 cross-linked cardiomyocytes compared with 31 ± 7% of control cardiomyocytes (P < 0.05). As a result, ICAM-1 cross-linking increased the duration of the Ca2+ transient (Fig. 8A). The slope over the most linear middle 50% of the decay curve was decreased to 1.53 ± 0.14 x 10–4 in ICAM-1 cross-linked cardiomyocytes from 2.94 ± 0.24 x 10–4 in control cardiomyocytes (P < 0.0001). Heterogeneity of Ca2+ release increased in the ICAM-1 cross-linked group as indicated by an increase in relative dispersion in ICAM-1 cross-linked cardiomyocytes (Fig. 8B). Total Ca2+ release (integral of Ca2+ release in Fig. 8A) did not differ significantly in ICAM-1 cross-linked cardiomyocytes vs. controls.



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  		Fig. 8. A: Ca2+ release and reuptake in control cardiomyocytes is rapid, whereas Ca2+ release in ICAM-1 cross-linked cardiomyocytes is much slower, with a wave of Ca2+ release moving along the length of the cardiomyocyte. B: average results for control (n = 37, solid line) and ICAM-1 cross-linked (n = 67, dashed line) cardiomyocytes, demonstrating increased dispersion, or heterogeneity, of Ca2+ release in ICAM-1 cross-linked cardiomyocytes. Coefficient of variation is particularly increased in ICAM-1 cross-linked cardiomyocytes for the first 1.5 s after cardiomyocyte electrical stimulation.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The key finding of this study is that ICAM-1 binding decreases cardiomyocyte contractility. ICAM-1 binding by freshly isolated PMNs and by PMNs fixed in paraformaldehyde or glutaraldehyde and ICAM-1 binding using cross-linking antibodies caused a significant decrease in cardiomyocyte contractility. The actin cytoskeleton plays a central role. Evidence for this includes increased immunofluorescent staining of cortical actin cytoskeleton-associated FAK (2 µm below the cell surface) after ICAM-1 cross-linking. The decrease in cardiomyocyte contractility due to ICAM-1 cross-linking could be prevented by cytochalasin D and, using an alternative strategy, by latrunculin A. Furthermore, inhibition of the cytoskeleton-associated Rho-kinase pathway also prevented decreased contractility due to ICAM-1 cross-linking. The ultimate mechanism of loss of contractility due to ICAM-1 binding appears to be loss of the normal coordination of intracellular Ca2+ release. Even though total intracellular Ca2+ release during a single contraction did not change significantly, the release did not occur in the normal coordinated manner throughout the entire cell over a short period of time, so fractional shortening decreased.

ICAM-1 expression on activated cardiomyocytes (12, 28, 48) plays an important role in ICAM-1-CD18-dependent adhesion of PMNs to cardiomyocytes (16, 48, 53). In a whole animal model, antibody to CD18 protects against myocardial damage and dysfunction after ischemia-reperfusion (33) and reduces PMN adherence within the heart. ICAM-1- and CD18-deficient mice demonstrate a marked reduction in PMN accumulation and myocardial necrosis after acute ischemia-reperfusion (38). Activated PMNs in vitro can cause dysfunction and kill cardiomyocytes within minutes via production of reactive oxygen intermediates (16, 41). However, this short time course and the cardiomyocyte necrosis caused by highly activated PMNs are not fully consistent with in vivo observations of the prolonged time course of ischemia-reperfusion-induced myocardial stunning (3) and sepsis (24, 25, 39, 40). It is useful to note that PMN adhesion to human umbilical vein endothelial cells after anoxia-reoxygenation demonstrates early effects (minutes) mediated by reactive oxygen intermediates and late effects (hours) involving other pathways (27) including the actin cytoskeleton (44). In these cells, PMN-induced cytoskeletal changes are inhibited by antibodies to ICAM-1 (51). Thus our results are consistent with, and extend, previous studies.

This novel mechanism of decreased cardiomyocyte contractility implicates the cytoskeleton in maintaining normal coordination of intracellular Ca2+ release and, hence, normal contractility. Ca2+-induced Ca2+ release from the sarcoplasmic reticulum triggered by Ca2+ influx via dihydropyridine receptors is the main regulator of cardiomyocyte contractility (4, 29). The novel finding that impaired coordination of Ca2+-induced Ca2+ release is an additional mechanism regulating cardiomyocyte contractility may have broad implications in myocardial processes involving inflammation and upregulation of ICAM-1 expression on cardiomyocytes. It may play a role in disease processes that involve leukocyte infiltration in the heart, including ischemia reperfusion, inflammatory cardiomyopathy, and septic myocardial dysfunction. Furthermore, disease and repair processes that alter extracellular matrix constituents so that increased ICAM binding occurs may also be implicated. For example, changing extracellular matrix composition and ICAM-1 expression also occur during normal embryogenesis (14, 34, 43).

PMNs and macrophages have previously been shown to bind ICAM-1 expressed on cardiomyocytes (16, 47, 48). In many of the studies of the interaction between highly activated PMNs and cardiomyocytes, ICAM-1 mediates binding and activated neutrophils cause cardiomyocyte cell death by release of reactive oxygen intermediates. The effect of nonactivated neutrophils and ICAM-1 binding by itself has not previously been examined. However, Simms and Walley (47) reported that ICAM-1 mediated the effects of macrophage/monocyte binding on decreased cardiomyocyte contractility. Recently, Raeburn et al. (42) reported that ICAM-1 and VCAM-1 mediate endotoxemic myocardial dysfunction independent of neutrophil accumulation in the myocardium. Although the earlier results implicate ICAM-1 largely as a mediator of tethering of cytotoxic neutrophils to cardiomyocytes (16, 48), these recent studies (42, 47) suggest a potential alternative role for ICAM-1 binding.

Our present results identify and illuminate this potential alternative role. Binding of freshly isolated PMNs to cardiomyocytes causes a decrease in contractility without cardiomyocyte cell death, presumably because the PMNs are not highly activated and releasing reactive oxygen intermediates and other damaging mediators. To confirm this and to exclude the possibility that PMN release damages mediators as a result of PMN activation during the isolation procedure, we repeated these experiments in paraformaldehyde- and glutaraldehyde-fixed PMNs. Surprisingly, even when PMNs are killed and fixed in two different ways to prevent further activation and release of mediators, PMNs cause a dose-dependent decrease in cardiomyocyte contractility (Fig. 3). The importance of ICAM-1 binding by itself in causing decreased contractility was then confirmed in a leukocyte-free condition where ICAM-1 cross-linking antibodies were applied, which results in a decrease in cardiomyocyte contractility.

This decrease in contractility clearly involves the cortical actin cytoskeleton. First, utilizing three-dimensional reconstruction techniques to examine the cortical region of cardiomyocytes, we found that cytoskeleton-associated FAK expression increases after ICAM-1 binding. Furthermore, inhibition of normal cytoskeletal function using cytochalasin D or latrunculin A prevented ICAM-1 binding-induced decreases in cardiomyocyte contractility. Finally, inhibition of the actin cytoskeleton-associated Rho-kinase signaling pathway also prevented ICAM-1 binding-induced decrease in contractility.

We found that the decrease in contractility was due to loss of simultaneity of intracellular Ca2+ release. One key coordinating mechanism of Ca2+-induced Ca2+ release is colocalization of dihydropyridine receptors on external or t tubule sarcolemma with ryanodine receptors on the sarcoplasmic reticulum (45). This geometric arrangement is instrumental in allowing the relatively small intracellular Ca2+ flux introduced via dihydropyridine receptors to trigger Ca2+-induced Ca2+ release by ryanodine receptors. We postulate that disruption of this coordinating mechanism would result in increased heterogeneity of Ca2+ release by the sarcoplasmic reticulum. That is, if dihydropyridine receptors do not cause coordinated induction of Ca2+-induced Ca2+ release by ryanodine receptors, then simultaneous contraction throughout the cardiomyocyte would be impaired. In the absence of normal coupling, a stimulus sufficient to cause local sarcoplasmic release of Ca2+ would then cause further Ca2+-induced Ca2+ release by adjacent sarcoplasmic reticulum ryanodine receptors. If true, this would take the form of a wave of contraction traveling from the initial site along the cardiomyocyte. Indeed, this is what we observed with ICAM-1 cross-linking (Fig. 8). Thus we think that it is reasonable to postulate that the actin cytoskeleton plays a key role in maintaining coordination of cardiomyocyte contractility, possibly by contributing to maintenance of normal colocalization of dihydropyridine receptors and ryanodine receptors.

Identification of this novel pathway, which may contribute to myocardial dysfunction during intramyocardial inflammation (e.g., after ischemia-reperfusion, in inflammatory cardiomyopathy, during orthotopic heart transplant rejection, and during sepsis-induced myocardial dysfunction), is clearly only one of many pathways involved. Indeed, ICAM-1 binding may be a component of some of these alternative pathways such as apoptosis (35) and reactive oxygen intermediate production (26).

It is interesting to speculate when and why such a mechanistic pathway may confer an evolutionary advantage. Is this mechanism of regulation of cardiomyocyte contractility just pathological loss of normal coordination, or is there some benefit? This novel pathway would be evoked during inflammation and subsequent resolution and repair or, conceivably, even during embryogenesis. We speculate that it may be beneficial to downregulate contractility for a period of time during these processes. This ICAM-1- and cytoskeleton-mediated pathway could provide this mechanism.

In summary, we have identified a novel pathway and mechanism of regulation of cardiomyocyte contractility. Although we have demonstrated that this pathway plays a role in neutrophil-cardiomyocyte interaction, it may play a role in the interaction of the cardiomyocyte with other inflammatory cells and potentially even with the extracellular matrix. Thus this novel mechanism of decreased contractility may have much broader implications.


   GRANTS
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by the Canadian Institutes of Health Research. K. R. Walley is a Distinguished Scholar of the Michael Smith Foundation for Health Research. D. R. Dorscheid is a Parker B. Francis Foundation Scholar and Michael Smith Foundation for Health Research Scholar.


   ACKNOWLEDGMENTS
 
The authors thank Yinjing Wang and Thoma Kareco for technical expertise and contributions in the preparation of the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. R. Walley, UBC, St. Paul's Hospital, 1081 Burrard St., Vancouver, BC, Canada V6Z 1Y6 (E-mail: kwalley{at}mrl.ubc.ca).

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. Section 1734 solely to indicate this fact.


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