Laboratory Investigation
Targeted Gene Silencing of Tumor Necrosis Factor Attenuates the Negative Inotropic Effects of Lipopoly saccharide in Isolated Contracting Cardiac Myocytes
R.S. Ramabadran, MD, PhD Amanda Chancey, PhD Jesus G. Vallejo, MD Philip M. Barger, MD Natarajan Sivasubramanian, PhD Douglas L. Mann, MD
Key words: Animals; cats; endotoxemia; myocytes, cardiac; lipopolysaccharides; myocardial contraction; RNA, double-stranded; RNA, messenger; toll-like receptor 4; tumor necrosis factor-alpha From: Winters Center for Heart Failure Research (Drs. Barger, Chancey, Mann, Ramabadran, and Sivasubramanian), Section of Cardiology, Department of Medicine, Baylor College of Medicine and the Texas Heart Institute at St. Luke’s Episcopal Hospital; and Section of Pediatric Infectious Disease (Dr. Vallejo), Texas Children’s Hospital, Baylor College of Medicine; Houston, Texas 77030 This research was supported by research funds from the National Institutes of Health (P50 HL-O6H and RO1 HL58081, and RO1 HL73017). Address for reprints: Douglas L. Mann, MD, BCM620, FC 9.83, 1709 Dryden Road, Houston, TX 77030
Bacterial endotoxin (lipopolysaccharide) depresses cardiovascular function; however, the mediators and signaling pathways that are responsible for the negative inotropic effects of lipopolysaccharide are not fully known. We used RNA interference to determine the relative role of tumor necrosis factor with respect to mediating the negative inotropic effects of lipopolysaccharide in isolated cardiac myocytes. Cardiac myocyte cultures were treated with lipopolysaccharide in the presence or absence of small interfering RNAs (siRNA) for tumor necrosis factor. We examined the effects of tumor necrosis factor siRNA on lipopolysaccharide-induced tumor necrosis factor messenger RNA (mRNA) and protein biosynthesis, as well as the negative inotropic effects of lipopolysaccharide in isolated contracting cardiac myocytes. Treatment of adult cardiac myocyte cultures with tumor necrosis factor siRNA significantly attenuated lipopolysaccharide-induced tumor necrosis factor mRNA and protein biosynthesis, whereas transfection with a double-stranded RNA that does not target mammalian mRNA had no effect. Pretreatment with tumor necrosis factor siRNA significantly attenuated, but did not abrogate, the lipopolysaccharide-induced decrease in sarcomere shortening in isolated contracting cardiac myocytes. In contrast, tumor necrosis factor siRNA had a comparatively smaller effect on improving sarcomere shortening once the negative inotropic effects of lipopolysaccharide were fully established. These results suggest that tumor necrosis factor plays an important upstream role in lipopolysaccharide-induced negative inotropic effects in isolated contracting cardiac myocytes and that other molecular mechanisms are responsible for the decrease in sarcomere shortening after sustained lipopolysaccharide signaling. (Tex Heart Inst J 2008;35(1):16-21)
acterial endotoxin (lipopolysaccharide [LPS]) triggers the release of a cascade of endogenous mediators and induces hypotension, multiorgan failure, and death from sepsis and septic shock.1,2 Although it has long been recognized that LPS depresses cardiovascular function, the precise signaling pathways that are responsible for mediating the negative inotropic effects of LPS have not been fully elucidated. Recent studies from this and other laboratories have shown that the deleterious effects of LPS are mediated, at least in part, through the toll-like receptor 4 (TLR4).3-6 Given that activation of the TLR4 pathway leads to the increased expression of pro-inflammatory cytokines, which in turn is sufficient to produce negative inotropic effects, we sought to use the recently developed technique of RNA interference (RNAi)7 to determine what role, if any, the pro-inflammatory cytokine tumor necrosis factor (TNF) plays in mediating the negative inotropic effects of LPS in isolated contracting cardiac myocytes. Here, we show for the 1st time that RNAi can be used to selectively knock down TNF gene expression in LPS-stimulated myocytes and that selective TNF-gene knockdown attenuates the deleterious effects of LPS in isolated contracting adult cardiac myocytes.
Materials and Methods Validation of siRNA Uptake in Cultured Cardiac Myocytes
E-mail:
[email protected] © 2008 by the Texas Heart ® Institute, Houston
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To confirm transfection of siRNA into cardiac myocytes, we used siGLO RISC-Free siRNA (Dharmacon, Inc., part of Thermo Fisher Scientific; Lafayette, Colo), a stable, non-targeting control siRNA labeled with a DY-547 (Cy-3 equivalent) fluorescent label that provides a reliable visual display of transfection success. For siRNA transfection into cardiac myocytes, we used GeneSilencer siRNA Transfection Re-
Lipolysaccharide and RNA Interference
Volume 35, Number 1, 2008
agent (Genlantis, a division of Gene Therapy System, Inc.; San Diego, Calif ), a proprietary cationic lipid formulation that provides high-efficiency siRNA transfer into mammalian cells. Briefly, freshly isolated feline cardiac myocytes were plated at a density of 8 × 105 cells per well (~50%–70% confluence) on laminin-coated 6-well plates that contained phenol-free M199 medium (GIBCO, Invitrogen Corporation; Carlsbad, Calif ) supplemented with insulin/transferrin/selenium (cellgro, Mediatech, Inc.; Manassas, Va) and 0.1% human serum albumin (Sigma-Aldrich; St. Louis, Mo). Cell cultures were incubated overnight at 37 °C at 95% O2, 5% CO2. Two hours before transfection with siRNA, the cell culture medium was changed and replaced with fresh medium M199 that was free of human serum albumin. At the time of transfection, 5 µL of siRNA and/ or 5 µL of GeneSilencer were added to each well, and the cells were incubated at 37 °C at 95% O2, 5% CO2 for 30 minutes. After 30 minutes, the cells were examined for the presence or absence of intracellular DY-547– labeled siRNA by means of an Olympus IX71 inverted microscope (Olympus America Inc.; Center Valley, Pa) at ×40 magnification, using a tetramethyl rhodamine iso-thiocyanate red filter set (540–565 nm). Generation of Feline dsRNA and siRNA
Total RNA from feline spleen was first isolated and used as a template to obtain complementary DNA (cDNA) for feline TNF. The cDNA for TNF was synthesized by means of the real-time polymerase chain reaction, using the sequence information from GenBank (Acc No: NM-001009835) and validated by DNA sequencing. In order to generate a template for synthesizing double-stranded RNA (dsRNA ) in vitro, a ~399-base pair cDNA template for feline TNF was further PCRsynthesized by use of chimeric oligonucleotides, which contained a T7 promoter sequence in addition to the TNF sequence ( 5´ TTA ATA CGA CTC ACT ATA GGG AGA AGG ATC ATC TTC TCG AAC TCC GAG TGA CAA GCC 3´ and 5´ TTA ATA CGA CTC ACT ATA GGG AGA CCC TTC AGC TTC GGG GTT TGC TGG 3´). The size of the cDNA template for TNF with opposing T7 promoters at the 5´ end of each strand is ~399 base pairs. The dsRNA for TNF was generated using the commercially available MEGAscript RNAi kit (Ambion, an Applied Biosystems business; Austin, Tex). The resultant dsRNA was subsequently diced and column-purified to obtain TNF siRNA by means of the Dicer siRNA Generation Kit (Genlantis), and stored at –20 °C. Effect of siRNA on LipopolysaccharideInduced Tumor Necrosis Factor Protein and mRNA Biosynthesis
Previously, we have shown that treatment of cultured adult cardiac myocytes with 10 to 100 µg/mL of LPS Texas Heart Institute Journal
results in a 3- to 5-fold increase in TNF levels that is maximal at 6 hours and declines thereafter.8 To determine the effects of TNF mRNA silencing on LPS-induced TNF biosynthesis, adult feline cardiac myocytes were isolated and cultured as described.9 On day 1 in culture, the myocytes were pretreated with TNF siRNA (500 ng/mL), nautilus dsRNA (25 µg/mL)—a nonmammalian dsRNA that does not target mammalian mRNA (negative control)—or diluent for 4 hours, followed by treatment with 25 µg/mL LPS (obtained from Escherichia coli Serotype 026:B6; Sigma-Aldrich) for 6 hours. The TNF biosynthesis was assayed in the cell culture media by use of an enzyme-linked immunosorbent assay (BioSource cytoscreen US Ultrasensitive, catalog #KHC3013; Invitrogen), with a detection range of 0.5 to 32 pg/mL, as described.8 Centricon microconcentrators (Amicon, Millipore; Billerica, Mass) were used to concentrate the cell culture medium by ultrafiltration through a low-adsorption, hydrophilic membrane with a cutoff value of 10,000 MW. For the analysis of LPS-induced TNF mRNA levels, cultured cardiac myocytes were treated for 4 hours either with TNF siRNA or nautilus dsRNA, as described above, or with diluent. The myocyte cultures were then stimulated with LPS for 90 minutes. The level of expression of TNF and L32 mRNA were determined using a custom-designed multiprobe riboquant ribonuclease protection assay (Pharmingen, BD Biosciences [San Jose, Calif], a segment of Becton, Dickinson and Company [Franklin Lakes, NJ]), as described.10 Effect of siRNA on LipopolysaccharideInduced Cardiac Myocyte Function
Sarcomere shortening of freshly isolated adult cardiac myocytes was evaluated by means of the IonOptix MyoCam system (IonOptix Corporation; Milton, Mass), as described.11 Freshly isolated myocyte cultures were pretreated for 4 hours with TNF siRNA (500 ng/mL), nautilus dsRNA (25 µg/mL), or diluent, and were then stimulated with 25 µg/mL LPS for 6 hours. Suspensions of cells were then placed in a chamber mounted on the stage of an inverted microscope and superfused with a buffer, containing (in mM) the following: HEPES, 20 (pH, 7.4); calcium chloride, 1; sodium chloride, 137; potassium chloride, 5.4; dextrose, 15; magnesium sulfate, 1.3; and sodium dihydrogenphosphate, 1.2. Experiments were conducted at 37 °C. The myocytes were field-stimulated at a frequency of 0.5 Hz; changes in sarcomere length during contraction were captured using IonOptix software. In parallel control experiments, myocytes were pre-incubated with etanercept (30 µg/mL), a soluble TNF antagonist, for 4 hours, and were then treated with 25 µg/mL LPS for 6 hours. To determine whether the effects of TNF siRNA were secondary to disruption of TNF signaling, TNF siRNA-treated cells (500 ng/mL) were stimulatLipolysaccharide and RNA Interference
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ed with TNF (200 U/mL) for 30 minutes, before the evaluation of cell motion. Finally, to determine whether TNF siRNA was sufficient to reverse LPS-induced defects in sarcomere shortening, cardiac myocyte cultures were treated with LPS (25 µg/mL) for 6 hours, and this was followed by treatment with TNF siRNA (500 ng/ mL) for 2 hours, before evaluation of cell motion.
30 minutes in the presence of GeneSilencer, a cationic lipid transfection reagent (Fig. 1D). The diluent and GeneSilencer alone did not show any background fluorescence (Figs. 1A and 1B). This figure is representative of 3 separate experiments.
Statistical Analysis
Effect of siRNA on LipopolysaccharideInduced Tumor Necrosis Factor Protein and mRNA Biosynthesis
Validation of siRNA Uptake in Isolated Cardiac Myocytes
To determine whether siRNA interference was sufficient to disrupt LPS-induced TNF biosynthesis, we examined TNF mRNA and protein biosynthesis in the presence and absence of TNF siRNA. The ribonuclease protection assay depicted in Figure 2A shows that TNF mRNA was detectable in LPS-treated cardiac myocytes, whereas pretreatment with TNF siRNA knocked down LPS-induced TNF mRNA expression. In contrast, transfection of cardiac myocytes with nautilus dsRNA had no effect on LPS-induced mRNA biosynthesis; L32 mRNA levels were not affected by TNF siRNA. Figure 2B shows that the baseline levels of su-
All values are expressed as mean ± SEM. One-way analysis of variance was used to test for differences in TNF concentrations and sarcomere shortening in the different experimental groups. Where appropriate, post hoc testing was performed using a Fisher exact test to detect differences between groups. Significant differences were said to exist at P
