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J Thorac Cardiovasc Surg 2005;129:1128-1136
© 2005 The American Association for Thoracic Surgery
Surgery for Congenital Heart Disease |
a Department of Cardiovascular Surgery, The Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada
c Department of Cell Biology, The Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada
b the Max Bell Research Centre, University Health Network, University of Toronto, Toronto, Ontario, Canada
d Medical Biophysics, Ontario Cancer Institute, Toronto, Ontario, Canada
Read at the Eighty-fourth Annual Meeting of The American Association for Thoracic Surgery, Toronto, Ontario, Canada, April 25–28, 2004.
Received for publication April 28, 2004; revisions received September 28, 2004; accepted for publication November 30, 2004. * Address for reprints: John G. Coles, MD, Division of Cardiovascular Surgery, Hospital for Sick Children, 555 University Ave, Toronto, Ontario M5G 1X8, Canada (E-mail: john.coles{at}sickkids.ca).
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Abstract |
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METHODS: Primary cultures of human fetal cardiac myocytes (gestational age, 15–20 weeks) were exposed to simulated ischemia and reperfusion in vitro by using a simulated ischemic buffer under anoxic conditions. Total RNA from treated and baseline cells were isolated, reverse transcribed, and labeled with Cy3 or Cy5 and hybridized to a human cDNA microarray for expression analysis. This analysis revealed a highly significant (false discovery rate, <3%) suppression of interleukin 6 transcript levels during the reperfusion phase confirmed by means of quantitative polymerase chain reaction (0.25 ± 0.11-fold). Interleukin 6 signaling during ischemia and reperfusion was assessed at the protein expression level by means of Western measurements of interleukin 6 receptor, the signaling subunit of the interleukin 6 receptor complex (gp130), and signal transducer of activated transcription 3. Posttranslational changes in the protein kinase B signaling pathway were determined on the basis of the phosphorylation status of protein kinase B, mitogen-activated protein kinase, and glycogen synthase kinase 3ß. The effect of suppression of a prohypertrophic kinase, integrin-linked kinase, with short-interfering RNA was determined in an ischemia and reperfusion-stressed neonatal rat cardiac myocyte model. Endogenous secretion of interleukin 6 protein in culture supernatants was measured by enzyme-linked immunosorbent assay.
RESULTS: Human fetal cardiac myocytes exhibited a significantly lower rate of apoptosis induction during ischemia and reperfusion and after exposure to staurosporine and recombinant interleukin 6 compared with that observed in neonatal rat cardiac myocytes (P < .05 for all comparisons, analysis of variance). Exposure to exogenously added recombinant interleukin 6 increased the apoptotic rate in both rat and human fetal cardiac myocytes (P < .05). Short-interfering RNA-mediated suppression of integrin-linked kinase, a prohypertrophy upstream kinase regulating protein kinase B and glycogen synthase kinase 3ß phosphorylation, was cytoprotective against ischemia and reperfusion-induced apoptosis in neonatal rat cardiac myocytes (P < .05).
CONCLUSIONS: Human fetal cardiac myocytes exhibit a uniquely adaptive transcriptional response to ischemia and reperfusion that is associated with an apoptosis-resistant phenotype. The stress-inducible fetal cardiac myocyte gene repertoire is a useful platform for identification of targets relevant to the mitigation of cardiac ischemic injury and highlights a novel avenue involving interleukin 6 modulation for preventing the cardiac myocyte injury associated with ischemia and reperfusion.
The idea that interleukin (IL) 6 pathway activation adversely affects cardiac function is solidly supported by clinical studies indicating that IL-6 and its specific receptor (IL-6R
) and the 130-kd glycoprotein signaling subunit of the IL-6 receptor, gp130,5 are upregulated at the mRNA and protein levels in the myocardium in patients with advanced heart failure in comparison with a control group6 and by the large increases in IL-6 plasma concentration that occur during cardiopulmonary bypass.7 These studies, however, do not differentiate between the inference that cardiac stress engenders endogenous release of IL-6 as a protective response and the diametrically opposed viewpoint that IL-6 is per se causative of cardiac damage.
Cellular responses to IL-6 are elicited by binding of soluble IL-6 cytokine to IL-6R. The signaling capacity of the IL-6/IL-6R complex is governed by the recruitment of 2 gp130 subunits to the activated, multisubunit receptor complex. Activation of the IL-6 receptor-ligand complex is dependent on the recruitment and dimerization of gp130,8 which triggers activation of several collateral signaling pathways, including the Janus kinase/signal transducer and activator of transcription (JAK/STAT), Ras/mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3–kinase (PI3-K)–dependent pathway involving sequential phosphorylation of protein kinase B (PKB/Akt) and glycogen synthase kinase 3ß (GSK-3ß).9 Thus the IL-6 receptor complex consists of a ligand-binding molecule (IL-6R) and a signaling subunit, gp130, which provides a rapid membrane-to-nucleus signaling system regulating inflammatory gene expression.
We show here in expression-profiling experiments that human fetal cardiac myocytes (HFCMs) exposed to simulated ischemia with reperfusion (I/R) or without reperfusion exhibit a uniquely adaptive transcriptional response. The fetal response includes a limited number of functional clusters dominated by predicted anti-inflammatory properties, featuring repression of the IL-6 signaling evident at both the mRNA and protein expression levels during reperfusion-mediated stress. These data provide a plausible and therapeutically important explanation for the innately apoptosis-resistant HFCM phenotype.
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Methods |
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HFCMs and NRCMs were exposed to simulated I/R or ischemia without reperfusion in vitro for the indicated time intervals by using ischemic buffer and anoxic conditions, as previously described.11 To simulate ischemia, the cultures were washed twice with phosphate-buffered saline and transferred to a pH 6.5 simulated ischemia solution containing, in 100 mL of deionized water, 0.8 g of NaCl, 0.119 g of N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, 0.164 g of 2-deoxyglucose, 0.89 mL of KCl, 0.472 mL of CaCl2, 0.238 mL of MgCl2, and 0.135 mL of DL-lactic acid saturated in 100% N2 for 1 hour, followed by addition of 0.1 g of bovine serum albumin. Cultures were placed in an airtight container at 37°C with 100% N2 inflow for periods ranging from 2 to 24 hours and subsequently washed twice with phosphate-buffered saline before harvesting. Cardiomyocyte cultures were re-exposed to the control normoxic serum-free medium to simulate reperfusion. Control cardiomyocyte cultures were maintained under normoxic conditions at 37°C until analysis. Thus ischemia is simulated by a combination of acidic pH, interference with glucose metabolism, and anoxia, and reperfusion is simulated by means of re-exposure to normoxia, which has been shown to provoke extensive apoptosis in similar models12 and is herein referred to as simulated I/R.
Microarray gene expression analysis
RNA isolation, fluorescence labeling of cDNA, hybridization to spotted arrays containing 15,264 sequence-verified cDNA clones, and quantitative fluorescence scanning of gene expression intensity were performed at the University of Toronto Health Network Microarray Centre (www.microarray.ca), as previously reported by us13 and others (for a list of publications, see http://www.microarrays.ca/about/pub.html). Significance of changes in sequential gene expression in HFCMs exposed to I/R (at control, 4 hours of ischemia, and 4 hours of ischemia plus 4 hours of reperfusion) were determined by means of repeated permutation of MIAME-compliant (www.mged.org) data with Significance Analysis for Microarray.14 Results from the Significance Analysis for Microarray analysis were visualized as hierarchic clusters in Gene Traffic (www.iobion.com), and significant genes were classified by their differential response to ischemia, reperfusion, or both. The results shown in Table 1 and Figure 1 are based on 2 biologic and 2 technical (array) replicates at each indicated time point, with a false discovery rate indicative of the statistical risk of incorrect identification of differentially expressed genes set to less than 3%.15
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Western blot analysis
Fetal cardiomyocyte extracts containing 20 µg of protein were subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis with 10% polyacrylamide gel and transferred onto Immobilon-P transfer membranes (Millipore). Analysis was performed with polyclonal PKB antibody (Transduction Laboratories), polyclonal serine 437 catalytically active, phosphorylation-specific PKB antibody (Cell Signaling Technology), polyclonal integrin-linked kinase (ILK) antibody (Upstate Biotechnology), and anti-IL-6 receptor (IL-6R) and anti-gp130 antibodies (Santa Cruz Biotech). Monoclonal antibodies used for the determination of total and phosphorylated GSK-3ß protein levels were from Biosource; total and phosphorylated (Py705) STAT-3, (Thr202/Tyr204) MAPK42/44, and stress-activated protein kinase (SAPK-Thr183/Pyr185) monoclonal antibodies were from Cell Signaling.
IL-6 measurements
IL-6 concentrations in the culture supernatants were determined by using an enzyme-linked immunosorbent assay kit according to the manufacturer’s instructions (Diaclone).16 The absorbance at 450 nm was measured, and concentrations were determined by means of interpolation of a standard calibration curve. The lower limit of detection of IL-6 was 0.78 pg/mL. Human recombinant IL-6 was from Sigma (1–1395).
Measurement of apoptosis
Apoptosis of variously treated cardiomyocytes was determined on the basis of nuclear condensation with Hoechst staining.17 Cardiomyocytes were stained with 1 µg/mL Hoescht 33342 trihydrochloride trihydrate (Molecular Probes) for detection of apoptotic nuclei. Dishes were analyzed at 20x magnification by using a Leica inverted deconvolution microscope with a coupled camera. Apoptotic cells were identified by their increased fluorescence caused by chromatin condensation and pyknotic morphology. A minimum of 300 nuclei were counted per field, and each data point consisted of 4 randomly selected fields. The measurement of cardiomyocyte apoptosis with Hoechst staining was found to correlate with, but was more sensitive than, that based on TdT-mediated dUTP nick-end labeling labeling with the APO-BRDU kit and enumeration by means of flow cytometry (FACScan/CELL Quest system, BD Biosciences) and that based on Western measurement of cleaved caspase-3 Asp175 antibody (Cell Signaling). The percentage of apoptosis was determined as the ratio of apoptotic nuclei/total Hoechst-positive nuclei, and statistical comparisons were made with Openlab 3.1.5 software. Statistical evaluation of intervention and cell type effects relied on a paired t test or 1-way analysis of variance. Data are expressed as ± standard error of the mean.
Synthesis and transfection of ILK-specific short-interfering RNA molecules
Single-stranded short-interfering RNA (siRNA) were transcribed and annealed with a commercial kit, as outlined in the supplier’s manual (Silencer Kit, Ambion). The following sequences were used to construct ILK siRNA: ILK1, 50-AAGGGGACCACCCGCACTCGG-30; ILK2, 50-AAGGCACCAATTTCGTCGTGG-30; and ILK3, 50-AAGCTCAACGAGAATCACTCT-30.
The specificity of ILK siRNA targeting vector has been previously shown.18 GAPDH control siRNA was provided with the Silencer siRNA construction kit. Transient transfections of NRCMs were carried out using 6 µL of Lipofectin reagent (Invitrogen), according to the manufacturer’s instructions. To quantitate the extent of knockdown of ILK protein, horseradish peroxidase-conjugated immunoglobulin G was used as a secondary antibody, and ILK immunocomplexes were visualized with an enhanced chemiluminescence detection reagent (Amersham Pharmacia Biotech) and quantified by means of densitometry.
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The fetal cardiomyocyte is resistant to apoptotic stimuli
The rate of apoptosis measured with Hoechst staining, as shown in Figure 2, was significantly lower in the fetal cardiomyocytes (relative to that observed in neonatal rat-derived cardiomyocytes) in response to increasing duration of ischemia with or without of reperfusion (P < .05 for rat vs human cardiomyocytes, analysis of variance). Exogenous IL-6 (250 ng/mL) caused a similar approximately 3- to 4-fold increase in apoptosis that was maximal at 3 hours of exposure in both NRCMs (P = .012) and HFCMs (P = .034) during normoxia and resulted in a significant increase in the apoptotic rates in both cellular phenotypes after both 4 and 10 hours of ischemia (P < .05). IL-6–mediated fold increases in apoptotic rates were greater in NRCMSs compared with that in HFCMs (P = .035).
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in HFCMs is expressed at low levels under control conditions, increases during 4 hours of ischemia, and increases to still higher levels after 4 hours of reperfusion (Figure 3, A). STAT-3 is highly phosphorylated under control conditions, becomes almost completely dephosphorylated during ischemia, and is rephosphorylated to intermediate levels after reperfusion. Because STAT-3 is activated by the signal transducer gp130 subunit, the conspicuous decrease in gp130 levels during I/R is consistent with the correspondent dephosphorylation of STAT-3. Unexpectedly, the addition of IL-6 resulted in a decrease in the levels of IL-6R, gp130, and PY705-phosphorylated STAT-3 during ischemia and after reperfusion. This finding might reflect counterregulatory degradation of IL-6R after ligation by exogenously added soluble IL-6. There was a coincident dephosphorylation of GSK-3ß at Ser-9 during I/R, which represents an inhibitory modification for this classically antihypertrophic kinase.19 The addition of IL-6 increased the extent of GSK-3ß phosphorylation under control conditions and after reperfusion in line with the prohypertrophic properties of this cytokine.5 Taken together, these data indicate posttranslational inhibition of IL-6 signaling during I/R and accords with the corresponding observed decrease in IL-6 message levels (Table 1).
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ILK knockdown protects against cardiomyocyte stress-induced apoptosis
Data on the effects of ILK knockdown in protection against cardiomyocyte stress-induced apoptosis are shown in Figure 4. ILK is a novel prohypertrophic kinase that causes phosphorylation of PKB/Akt and GSK-3ß.18 We asked whether siRNA-mediated suppression of this proproliferative kinase, which should mimic the signaling effects observed in the fetal cardiomyocytes, could influence apoptotic threshold in the NRCMs during I/R. As shown in the insert of Figure 4, lipofectamine-mediated transfection of the ILK-specific siRNAs, but not the GAPDH siRNA, resulted in substantial (42%, P = .02) knockdown of ILK expression in NRCMs as determined by means of Western blot analysis at 72 hours after transduction. Exposure of ILK-silenced NRCMs to 4 hours of ischemia and 4 hours of reperfusion resulted in an approximate 50% decrease in the apoptosis rate in comparison with that seen in lipofectamine-only controls (P = .031).
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Discussion |
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Genome expression profiling in the fetal cardiomyocyte revealed a conspicuous repression, or lack of induction, of IL-6 transcription during ischemia and especially during reperfusion (58% of preischemic levels by means of microarray, 25% by means of qPCR). IL-6 is a multifunctional cytokine with proproliferative and prohypertrophic properties in the heart.23 Upregulation of serum24 and myocardial levels of pro-inflammatory cytokines (tumor necrosis factor , IL-1, and IL-6) have been reported in infants with tetralogy of Fallot,25 and increased IL-6 message is found in rat hearts undergoing I/R.26 However, the specific alterations in the IL-6 signaling pathway induced in the human cardiomyocyte during I/R are unknown, and it is unresolved in the literature as to whether a stress-induced increase in circulating IL-6 levels represents a cardiomyocyte-protective or cardiomyocyte-injurious response.
Our results indicate that the IL-6 pathway is inhibited at multiple levels of regulation in HFCMs in response to I/R, including gp130 receptor expression, dephosphorylation of cytoplasmic, IL-6–dependent kinases (STAT-3, PKB/Akt, and GSK-3ß), and IL-6 transcription. Reduced STAT-3 phosphorylation and IL-6 transcription could result from downregulation of gp130 expression because this represents the proximal signaling module of the IL-6 receptor complex,8,9 despite the finding of a concomitant increase in the IL-6R subunit during both the ischemic and reperfusion phases. The reason for downregulation of gp130 expression is unknown. Ischemia-induced disruption of membrane lipid rafts plausibly accounts for this finding because IL-6 receptor complex localization and STAT-3 signal transduction are raft dependent.27
The finding that IL-6 potentiates stress-induced cardiomyocyte apoptosis appears to conflict with previous reports demonstrating cardioprotective properties of this cytokine.5,28 It might be speculated, however, that the mitogenic and prohypertrophic state associated with IL-6 stimulation could be energetically unfavorable under conditions of severe oxygen deprivation preceding reoxygenation, especially because IL-6 signaling has been linked to the generation of reactive oxygen species.29 The use of unbiased genome profiling provides further support for the idea that the HFCMs acquire a quiescent phenotype in response to oxygen restriction, including the finding of a significant reduction in the expression of MAPK 1 (Table 1). Activation of the MAPK cascade promotes an activating phosphorylation of the nuclear factor for IL-6, also termed C/EBPß, a member of the CCAAT/enhancer-binding protein (c/EBP) family of transcription factors. Nuclear factor for IL-6 binds to a regulatory element in the IL-6 promoter that is essential for induction of IL-6 transcription after treatment with tumor necrosis factor and IL-1.30 Deinduction of the MAPK signaling cascade, resulting from both decreased transcript and protein phosphorylation levels observed in the HFCM, might serve the energetically beneficial purpose of dampening agonist-induced, proinflammatory, and proproliferative signaling during I/R. IL-6 signaling might involve PI3-K-dependent, as well as STAT-3 and MAPK, pathways.21 Our data also indicate that deactivation of the PI3-K-dependent kinase PKB/Akt occurs in the HFCMs during I/R, thereby nullifying potential activation of IL-6 signaling through this collateral pathway.
ILK is a prohypertrophic kinase that is regulated in a PI3-K-dependent manner after distinct signal inputs from integrins and growth factor receptor tyrosine kinases.18 ILK causes phosphorylation of PKB/Akt and GSK-3ß,18 posttranslational modifications that are diametrically opposite to those that were observed in HFCMs exposed to ischemic stress. Therefore we used siRNA-mediated silencing of ILK to recapitulate the fetal response in the more apoptosis-prone rat cardiomyocyte model. This manipulation revealed an antiapoptotic effect of ILK knockdown and provides additional indirect evidence that inhibition of prohypertrophic signaling might represent a cardioprotective strategy under conditions of severe oxygen deprivation.
The induction of the antioxidant gene metallothionein I observed in the fetal cardiomyocyte transcriptional profile during I/R suggests an additional explanation for the apoptosis resistance evident in this cellular phenotype because this antioxidant has been shown to directly potentiate antiapoptotic signal transduction, as well as to mitigate redox-mediated injury.31 The functional significance of other differentially expressed genes identified in this study (Table 1), either singly or in combinatorial permutations, will require further experimentation conducted in both vulnerable and apoptosis-resistant cardiomyocyte phenotypes involving gene-specific gain- and loss-of-function strategies.
Taken together, our data support the general conclusion that the HFCM represents a useful biosynthetic platform for the identification of innate cardioprotective responses at the molecular level. The adaptive stress response exemplified by the fetal cardiomyocyte implicates the IL-6 pathway as an important and therapeutically relevant arbiter of cellular survival.
Limitations to the study
Our data indicate that the isolated HFCM is more resistant to apoptosis induction after exposure to simulated I/R than that of the neonatal rat. The difference in stress-induced apoptotic threshold is explainable on the basis of either species-related or development stage-related differences inherent in these distinct cellular phenotypes. The elucidation of developmental stage-specific aspects of the human cardiomyocyte stress response would require a comparative analysis of human neonatal or adult cardiomyocytes with that of the fetal phenotype. The fact that postnatal human cardiomyocytes have an inherently greater susceptibility to apoptosis during in vitro culture renders such studies difficult and implies that cardiomyocyte stress resistance degrades with increasing maturation. Nevertheless, our study is directed to the investigation of IL-6 signaling during redox-induced stress in the apoptosis-resistant HFCM phenotype and does not specifically address the developmental aspects of this response.
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Dr Coles. We have not successfully cultured them for more than about 2 weeks.
Dr Chitwood. What would you think would happen at a month?
Dr Coles. Well, I think in culture you can have unanticipated effects, and sometimes the cells will dedifferentiate.
Dr Chitwood. So they might differentiate from being a cardiomyocyte and become something else.
Dr Coles. Correct. However, longer-term studies would be important and might reveal phenotypic differences unique to the fetal cell.
Dr Frank A. Pigula (Boston, Mass). John, that was fascinating. I think it would be particularly interesting to look at postnatal myocardial expression to see whether there is any difference between the two. And the only other question that comes to my mind is that these hearts, by virtue of how you obtain them, have been preconditioned to some degree, and I wonder whether that has any influence on the ultimate genotype and the expressions.
Dr Coles. Those are both fair points.
What we want to do is look at more apoptosis- vulnerable phenotypes, including, for example, cardiomyocytes derived from patients with congenital heart disease, such as tetralogy of Fallot.
Those cells are difficult to grow, however, which, I think, speaks to the fact that they are less resilient than the fetal phenotype. But I agree that if we can look at gain- and loss-of-function manipulations affecting the IL-6 pathway in cells such as tetralogy cells, this will help reinforce our findings regarding the importance of the IL-6 pathway. Preconditioning effects, if any, inherent in the cell-isolation procedure, would be difficult to measure but presumably would be factored out by comparison with more mature cellular phenotypes using the same protocol.
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and -
isoforms. Am J Physiol Heart Circ Physiol. 2001;281:H404-H410.This article has been cited by other articles:
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 S. Arab, I. E. Konstantinov, C. Boscarino, E. Cukerman, A. Mori, J. Li, P. P. Liu, A. N. Redington, and J. G. Coles Early gene expression profiles during intraoperative myocardial ischemia-reperfusion in cardiac surgery J. Thorac. Cardiovasc. Surg., July 1, 2007; 134(1): 74 - 81. [Abstract] [Full Text] [PDF]
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 H. Lu, P. W. M. Fedak, X. Dai, C. Du, Y.-Q. Zhou, M. Henkelman, P. S. Mongroo, A. Lau, H. Yamabi, A. Hinek, et al. Integrin-Linked Kinase Expression Is Elevated in Human Cardiac Hypertrophy and Induces Hypertrophy in Transgenic Mice Circulation, November 21, 2006; 114(21): 2271 - 2279. [Abstract] [Full Text] [PDF]
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