INTERNET SUPPLEMENT TO “Functional Significance of Hemodynamic Overload Induced Expression of Leukemia Inhibitory Factor In The Adult Mammalian"
ADDITIONAL METHODS
The following methodologies are intended as supplementary material to the manuscript
Regulation of LIF Expression in the Adult Heart Ex Vivo
LIF mRNA Biosynthesis. All myocardial biopsy samples were immediately frozen in liquid nitrogen. Total RNA was extracted from the myocardial samples using RNA-STAT (Tel-Test, Inc.), and was processed and hybridized using either a 570 bp Hind III/EcoR I fragment of human LIF (generously provided by Dr. Colin L. Stewart1) or a 0.5-kb Xba/Hind III fragment of human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [American Tissue Culture Collection (ATCC)]. The membranes were washed with standard saline citrate (2xSSC) and 0.5% sodium dodecyl sulfate (SDS) at 55 _C for 30 minutes, air dried, and then exposed to Kodak X-Omat A film at -70 _C with intensifying screens.
LIF Protein Biosynthesis: Western Blotting Myocardial extracts from hearts perfused at normal and elevated pressures were lysed at 4 _C in radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 10 mM Tris-HCl, pH7.4, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate, 1 mM EDTA) containing a cocktail of protease inhibitors (Boerhringer Mannheim). Equivalent amounts (20 g) of myocardial protein lysates were then loaded onto 12% SDS-polyacrylamide gels, electrophoretically separated, and transferred to nitrocellulose membranes as described.2,3 Western blotting analysis for LIF was performed using a polyclonal anti-LIF antibody (PharMingen, 1:1000 dilution) as the primary antibody and a horseradish peroxidase-conjugated secondary antibody (Amersham, 1:2000 dilution); the resulting protein levels were visualized by enhanced chemiluminescence (Amersham). The membranes were then washed and re-probed with an anti-actin monoclonal antibody (Amersham) to ensure equal loading of the lanes.
LIF Protein Biosynthesis: Bioassay. LIF bioactivity was examined in the superfusates from the buffer perfused hearts. For these studies a 5 ml sample of recirculating superfusate was collected, starting at time zero and for every 30 minutes thereafter, for a total of 180 minutes. All samples were immediately frozen at - 20 _C after collection. LIF bioactivity was determined by examining the effects of LIF on M1 cell proliferation (ATCC; TIB-192 ) as described,4 with the exception that cell number was determined using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium). The MTT assay was performed as we have described;5 recombinant human LIF was used as the standard.
Cellular Source for Myocardial LIF Production
LIF Production in Cardiac Myocytes Isolated from Pressure Overloaded Hearts. We examined the relative production of LIF mRNA and protein by myocyte and non-myocyte cell types isolated from pressure overloaded hearts, as we have described previously (see Supplement for details).6 Briefly, hemodynamic pressure overloading was performed continuously for 180 minutes. Immediately after pressure overloading, the hearts were enzymatically digested using techniques standard in this laboratory.7 Following enzymatic digestion of the heart the cells were rinsed with KHB and gently centrifuged (200 g) in order to separate the myocyte from non- myocyte cell types. The resultant cell pellet which contains > 95% cardiac myocytes, has < 2% contaminating fibroblasts8,9 and < 1% contaminating monocytes.10 The cell pellets and supernatant were then split into two portions and analyzed for LIF mRNA and protein production. Second, we examined LIF expression in cultured cardiac myocytes. Adult feline cardiac myocytes were isolated and maintained in culture as described previously.8 On the first day after isolation, the culture medium was changed and the myocyte cultures were stimulated with a concentration of TNF (200 U/ml) that is produced within the myocardium following hemodynamic overloading, 6 as well as endotoxin (125 g/ml), which is a potent inducer of a variety of pro-inflammatory cytokines. LIF mRNA expression was examined by Northern blot analysis after 3 hours of stimulation, whereas LIF protein production was examined by Western blot analysis after 6 hours of stimulation.
Functional Effects of LIF in Adult Cardiac Myocytes
To determine the functional consequences of LIF expression in the adult heart, we examined the effects of LIF on cardiac myocyte growth, cardiac myocyte viability (apoptosis) and cardiac myocyte contractility. For these studies we employed recombinant human LIF (2.3 x 104 U/g; R & D System), recombinant human IL-6 ( 1.1 x 105 IU/g; R & D System), and IL-6 receptor (R & D System).
Growth Stimulatory Effects of LIF . The methods for determining protein synthesis have been described in detail.11 Cardiac myocyte cultures (day 1) were labeled with 30 mCi/ml [3H]phenylalanine, in the absence or presence of LIF (0.01-10 ng/ml), or with IL-6 (100-4000 U/ml) and soluble IL-6 receptor (50 ng/ml). After 24 hours of stimulation, the cultures were harvested and the amount of [3H]phenylalanine incorporation into myocyte protein was determined. We examined the protein synthetic rate in cells stimulated with "conditioned medium", which was obtained from myocyte cultures which had been previously stimulated for 24 hours with either LIF (10 ng/ml) or diluent alone. To remove any LIF that might have remained in the conditioned medium, the medium was treated with 50 g/ml polyclonal anti- human LIF neutralizing antibody ( R & D System) and then centrifuged (10,000 g for 15 mins) to remove any LIF that might have remained in the conditioned medium. The myocyte cultures were then stimulated with the resultant supernatant .12 The methods for determining net actin and myosin heavy chain protein synthesis have been described in detail.11 Cardiac myocyte cultures (day 1) were radiolabeled with 30 mCi/ml [3H]phenylalanine for 72 hours in the presence or absence of LIF (10 ng/ml).11
Cytoprotective Effects of LIF: Assessment of Cardiac Myocyte Apoptosis using the DNA ligase technique. The extent of cardiac myocyte apoptosis was determined using a modification of the recently described in situ DNA ligation technique.13 Briefly, fixed cells were incubated overnight at room temperature with a ligation buffer (32.5 g/ml hairpin oligonucleotides , 15% polyethylene glycol , 250 U/ml T4 DNA ligase in 1x T4 DNA ligase buffer [Boehringer Mannheim]) in a humidified chamber. Biotin labeled hairpin oligonucleotides were synthesized as described,13 with the stems of the oligonucleotide ending in a double-stranded structure with a 3' protruding end that can be ligated with T4 DNA ligase to apoptotic double strand DNA breaks in cells. The loops of the oligonucleotide were modified by the attachment of biotin. The reaction was stopped by rinsing the slides x3 with H2O and then PBS. Apoptotic DNA strand breaks were detected using a fluorescein (FITC)-avidin conjugate (Vector Laboratories) that binds to the biotin labeled hairpin oligonucleotides. The extent of DNA labeling was determined by fluorescence microscopy (excitation/emission wavelength, 495/525 nm), by examining 5 randomly selected fields (at 200x magnification) for each myocyte culture. Rod and ball shaped cardiac myocytes containing at least one FITC labeled nucleus were identified as positive for apoptosis. Myocyte cultures that had been fixed and treated with DNase I served as the appropriate controls for each experiment. Final results were expressed as the percentage of apoptotic rod and ball shaped cells, as well as the number of apoptotic rod shaped cells.
Mechanism for the cytoprotective effect of LIF Cells were treated with LIF (0.1 - 10 ng/ml) for 5 minutes, after which the cells were washed with cold PBS and then lysed in SDS sample buffer (62.5 mM Tris-HCL, pH 6.8, 2% SDS, 1% glycerol, 50 mM DTT). The cell lysates were collected and the protein content determined using the BCA (bicinchoninic acid) assay.14 Equivalent amounts (40 g) of cell lysates were electrophoretically separated on 10% SDS-PAGE, and transferred to nitrocellulose membranes. Phosphorylated and total ERK1/ERK2 were detected by Western blot analysis, using a commercially available kit (PhosphoPlus p44/p42 MAP kinase, New England Biolab Inc) that employed a phospho-p44/42 MAP kinase antibody (1:500). Insofar as previous studies have suggested that tyrosine phosphorylation is necessary for STAT dimerization, and that serine phosphorylation is required for maximal transcriptional activity of STATs,15 we examined STAT3 phosphorylation on both tyrosine and serine residues. Tyrosine phosphorylation (Y705) of STAT3 was detected using a phospho-specific STAT3 primary antibody (1:500; New England Biolab Inc) and a secondary antibody that was conjugated to horseradish peroxidase. The membranes were washed and re- probed using a phospho-specific serine 727 (S727) STAT3 antibody (1 :500; New England Biolab Inc).
Cell motion assay. Previous studies have shown that ligands that signal through the gp130 pathway influence myocardial function,16 as well as alter calcium homeostasis. 17 Accordingly, we sought to determine whether LIF stimulation would affect cell motion of isolated cardiac myocytes. Freshly isolated feline cardiac myocytes were allowed to stabilize for 1 hour and were then treated with LIF (1, 10 ng/ml) for 30 and 60 mins at 37 _C. Cell motion was determined by video edge detection. 12 As additional control experiments we treated the myocytes with 10 nM isoproterenol (Sigma) or 200 U/ml tumor necrosis factor (Genzyme Corporation), in order to determine the ability of the myocytes to respond to positive and negative inotropic stimuli, respectively. Lastly, in order to determine whether LIF would blunt signaling through the -adrenergic receptor, we pretreated the myocyte cultures with LIF for 30 and 60 mins, and then stimulated the cells with 10 nM isoproterenol (Sigma).
ADDITIONAL RESULTS
The following experimental results are intended as supplementary material to the manuscript
Functional Effects of LIF in Adult Cardiac Myocytes
Growth Stimulatory Effects of LIF . Figure S1-A shows that stimulation with LIF (0.01-10 ng/ml) provoked a concentration-dependent increase in the rate of general protein synthesis in isolated adult cardiac myocytes. That is, treatment with 0.01 ng/ml LIF had no significant effect on protein synthesis, whereas stimulation of the myocyte cultures with > 0.1 ng/ml LIF resulted in a progressive increase in the rate of general protein synthesis (n = 5 cultures per LIF concentration). The specificity of the effects of LIF was shown by the studies wherein we used a neutralizing anti-LIF antibody to completely block the effects of LIF (n = 10 cultures). LIF conditioned media had no effect on protein synthesis (n = 13 cultures), suggesting that growth stimulatory effects of LIF were direct, as opposed to being mediated indirectly by the release of soluble autocrine/paracrine growth factors. Moreover, the maximal rate of protein synthesis with LIF (10 ng/ml) was greater than the maximal rate of protein synthesis observed with IL-6 (2000 U/ml + 50 ng/ml soluble IL-6 receptor). One-way ANOVA indicated that there were significant (p < 0.001) overall differences in protein synthesis in the LIF stimulated myocyte cultures; post-hoc analysis of variance testing (Tukey) indicated that LIF concentrations were significantly (p < 0.01) different from control values for concentrations of LIF > 0.1 ng/ml. Moreover, protein synthesis was significantly greater (p < 0.01) in LIF stimulated myocyte cultures when compared to the maximal degree of protein synthesis obtained by stimulating the myocyte cultures with IL-6 and IL-6sR. To determine whether LIF stimulation would lead to a net increase in sarcomeric protein synthesis, we examined net actin and myosin heavy chain protein synthesis in myocyte cultures that were continuously stimulated with LIF (10 ng/ml) for 72 hours. As shown in Figure S1-B, stimulation with LIF led to a significant 1.27 Å 0.1-fold increase (p < 0.01) in net actin protein synthesis (n = 15 cultures) and a significant 1.47 Å 0.1-fold increase (p < 0.005) in net myosin heavy chain synthesis (n = 15 cultures), when compared to diluent treated control cultures.
Effects of LIF on Cell Motion. Figure S1-A summarizes the results of the studies wherein we examined cell motion in cardiac myocytes that had been stimulated with LIF (1, 10 ng/ml) for 30 min and 60 mins. As shown, there was no significant change in cell shortening for the cells that were treated with either 1 or 10 ng/ml of LIF for 30 or 60 mins, whereas there was a significant (p < 0.01) ë30 % increase in cell shortening in the cells treated with isoproterenol and significant (p <0 .05) ë 15 % decrease in cell shortening in the cells treated with tumor necrosis factor (Figure S2-B). Finally, we did not observe any blunting of cell shortening for isoproterenol treated cells that had been pre-treated with LIF (S2-B). Taken together, these studies suggesting that short term treatment with LIF does not modulate cell motion in adult cardiac myocytes. One-way ANOVA indicated that there were no significant overall differences in cell shortening between groups (p > 0.05 for both) in cell shortening for the LIF stimulated cardiac myocytes. In contrast, post-hoc analysis of variance testing (Tukey) showed that there was a significant increase in cell shortening for the isoproterenol treated cells (p < 0.05) and a significant decrease in cell shortening for the TNF treated cells (p < 0.05); there was, however, no significant difference in the extent of cell shortening when isoproterenol treated cells were compared in the presence and absence of LIF (p > 0.05) .
REFERENCES FOR SUPPLEMENT
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