1Genzyme Corporation, Framingham, Massachusetts; and 2Department of Internal Medicine, Jikei University School of Medicine, Tokyo, Japan
Submitted 30 July 2004 ; accepted in final form 12 October 2004
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
ischemic preconditioning; myocardial protection
A critical component of myocardial ischemia is hypoxia, which triggers a wide range of profound cellular responses, including rapid posttranslational modifications of proteins and regulation of gene expression. The paradigm for gene regulation in response to hypoxia involves oxygen sensing followed by a series of signal transduction mechanisms that lead to induction or repression of transcription and changes in mRNA stability or the rate of translation (10). Hypoxia-inducible factor-1 (HIF-1), a heterodimeric basic helix-loop-helix-PAS (bHLH-PAS) domain transcription factor, mediates the transcriptional activation of hypoxia-responsive genes (32). HIF-1 is composed of two subunits, HIF-1 and HIF-1
(aryl hydrocarbon nuclear translocator). Whereas HIF-1
is constitutively expressed, the stability, DNA binding capability, and transcriptional activity of the HIF-
subunit are directly controlled by the intracellular oxygen concentration (9, 12, 32). Recent studies have demonstrated that protection of rat brain and retinal tissues by prior exposure to an episode of sublethal hypoxia is associated with elevated levels of the HIF-1
protein (6, 7, 14, 30). In the neonatal rat brain HIF-1 target genes including vascular endothelial growth factor (VEGF) and Glut-1 have been shown to be upregulated by preconditioning stimulus. These results imply that HIF-1 may play a role in preconditioning of neural tissues. Indeed, several mediators and other cardioprotective genes including iNOS, HSP70, erythropoietin (EPO), and VEGF are also known hypoxia-responsive or HIF-1 target genes. Recently, we demonstrated (34, 37) that expression of HIF-1
/VP16, a constitutively stable hybrid of HIF-1
and herpes simplex virus (HSV) VP16 protein, mimics the angiogenic response to hypoxia in vitro and in vivo by inducing multiple growth factors. In human fetal cardiac cells, adenovirus-mediated expression of HIF-1
/VP16 upregulated several known HIF-1-inducible genes, including VEGF, Glut-1, Glut-3, and glycolytic enzymes (18). Insulin growth factor binding protein-3 (IGFBP-3), gp130, survivin, and TNF-
-inducible protein were also upregulated (18). These genes may directly or indirectly promote cell survival by various mechanisms. However, it is not clear whether adenovirus-mediated expression of HIF-1
/VP16 is capable of protecting cardiomyocytes against ischemia-reperfusion injury.
In this study, we investigated the effects of adenovirus-mediated expression of hybrid forms of HIF-1 on ischemia-reperfusion injury and the mRNA levels of known preconditioning mediators. We also measured the cellular protein levels of HIF-1
and the mRNA levels of several HIF-1 target genes in a cultured neonatal rat cardiomyocyte model of preconditioning (2, 13, 23, 28, 35, 38). Our results suggest that expression of a constitutively stable hybrid HIF-1
protects cultured neonatal cardiomyocytes against simulated ischemia-reperfusion injury by inducing multiple protective genes.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell culture. The investigation conforms to the NIH Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 86-23). Neonatal rat ventricular cardiomyocytes were isolated and cultured as described previously (11). Briefly, the cells were seeded at a density of 2.0 x 105 cells/cm2 on rat type I collagen-coated dishes and maintained in Dulbecco's modified Eagle's medium supplemented with 10% (vol/vol) fetal bovine serum for 4872 h before use in the studies.
Effect of preconditioning on cardiomyocyte death. As described previously (23, 35), preconditioning was created by placing the cultured neonatal rat ventricular cardiomyocytes in a hypoxic chamber (1% O2, 5% CO2; 37°C) for 3 h and then returned to control conditions (21% O2, 5% CO2; 37°C) for 24 h, followed by simulated lethal ischemia (depletion of serum and glucose and hypoxia) for 2 h. The cardiomyocyte cultures were then maintained under control conditions for an additional 15 h. The cellular level of 3-(4,5-dimethylthiazol-2-yl-5)-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), indicative of the mitochondrial function in living cells and cell viability, and lactate dehydrogenase (LDH) levels in the culture medium were measured with the CellTiter96 Aqueous One kit (Promega) and the TOX-7 assay kit (Sigma), respectively (11).
Measurement of mRNA levels. The mRNA levels were measured with TaqMan 5' nuclease fluorigenic quantitative PCR, as described previously (5). Briefly, primers and probes (Table 1) were designed and synthesized according to ABI-Perkin Elmer guidelines (Foster City, CA) and synthesized by Qiagen-Operon (Alameda, CA). Standard curves for each gene of interest were performed in duplicate. The mRNA levels of the gene of interest were normalized to 18S rRNA and expressed as fold increase over control.
|
Statistical analysis. Data are expressed as means ± SE. The number of samples examined is indicated by n. Data were analyzed by ANOVA, followed by a modified Student's t-test. A probability value of <0.05 was considered statistically significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
In response to cellular hypoxia, cardiomyocytes adapt to consuming less oxygen by shifting ATP production from mitochondrial fatty acid -oxidation to glycolysis (3, 10). Glucose-insulin-potassium infusion improves the survival of patients with ischemic heart disease, partially because of enhanced glucose uptake and glycolysis by the ischemic myocardium (1). Studies in transgenic mice overexpressing HSP70 and in rats that had received HSP70 expression vectors also indicate that this protein protects myocardium against ischemia-reperfusion injury (17, 24, 26, 29, 31). Recently we reported (18) that in human fetal cardiac cells, adenovirus-mediated expression of HIF-1
/VP16 upregulated VEGF, Glut-1, Glut-3, IGFBP-3, gp130, survivin, TNF-
-inducible protein, and glycolytic enzymes. In the present study, we observed that adenovirus-mediated expression of either HIF-1
/VP16 or HIF-1
/NF-
B significantly increased mRNA levels of Glut-1, Glut-4, and HSP70. Upregulation of these genes may partially explain the protection of cultured neonatal cardiomyocytes against simulated ischemia-reperfusion by adenovirus-mediated expression of either HIF-1
/VP16 or HIF-1
/NF-
B. It is known that HIF-1 upregulates expression of glucose transporters and glycolytic enzymes at the transcriptional level by binding to the hypoxia-responsive elements in the promoter of these genes (32). It remains to be determined whether HIF-1
/VP16 or HIF-1
/NF-
B activates HSP70 transcription by binding to its promoter.
NO plays a dual role in the pathophysiology of the late phase of preconditioning (8, 23). Enhanced biosynthesis of NO by eNOS is essential to trigger the late phase of ischemia-induced or exercise-induced preconditioning. Enhanced NO production by iNOS is obligatorily required to mediate the antistunning and anti-infarct actions of the late phase of preconditioning elicited by different stimuli. The generation of NO associated with an ischemic stress initiates a cascade of events that involves the activation of several kinases and transcriptional factors and culminates in the upregulation of the iNOS gene.
It is known that hypoxic activation of iNOS transcription is mediated by HIF in cardiomyocytes (19, 25). The increase of iNOS mRNA levels by the infection of cultured neonatal cardiomyocytes with Ad2/HIF-1/VP16 or Ad2/HIF-1
/NF-
B implies that the upregulation of this protein is an important protective mechanism by expression of a constitutively stable hybrid HIF-1
in this particular experimental setting. Together, our results suggest that expression of a constitutively stable hybrid HIF-1
protected cultured neonatal cardiomyocytes against simulated ischemia-reperfusion injury by inducing multiple protective genes.
It has been reported that ischemic preconditioning upregulates VEGF expression and neovascularization, which in return reduces infarct size on subsequent lethal ischemia (20). HIF-1 activates VEGF transcription by binding to the hypoxia-responsive element of its promoter (32). Recently we reported (5, 18, 34) that HIF-1/VP16 induced VEGF expression in various cells including rat ventricular myocytes. In the present study, we demonstrated that adenovirus-mediated expression of either HIF-1
/VP16 or HIF-1
/NF-
B significantly increased VEGF mRNA levels, suggesting that the upregulation of VEGF expression by constitutively stable hybrid forms of HIF-1
is independent of the transactivation domain. It is well known that administration of selective adenosine receptor (A1 and A3) agonists or pharmacological NO donors in the absence of ischemia induces a delayed protective effect on cardiomyocytes against lethal ischemia-reperfusion that is similar to that observed during ischemic preconditioning in vivo, presumably through protein kinases/AP-1- and/or NF-
B-mediated gene expression (8). Adenosine receptor agonists and NO can also increase myocardial oxygen availability through their vasodilator effect. Whether VEGF can directly act on cardiomyocytes and exhibit protective effects remains to be determined. One may speculate that VEGF expression induced by a constitutively stable hybrid form of HIF-1
may improve myocardial perfusion in vivo through increased vascular permeability and angiogenesis.
Involvement of HIF-1 in protection of cultured neonatal cardiomyocytes by preconditioning.
The late phase of preconditioning requires simultaneous activation of multiple mediator/effector genes (8). It has been suggested that AP-1 and NF-B may mediate the transcriptional activation of several mediators that are involved in ischemic preconditioning (22, 36). It is also known that transcription of iNOS, an important mediator/effector of myocardial preconditioning (8), is directly activated by HIF-1 in response to hypoxia (9, 12, 19, 32). Protection of rat brain and retinal tissues against subsequent lethal ischemia by prior exposure to sublethal hypoxia is associated with elevated levels of the HIF-1
protein (6, 7, 14). In this study, we demonstrated that in isolated neonatal rat cardiomyocytes the protein levels of HIF-1
were elevated by preconditioning. The mRNA levels of several hypoxia-responsive genes including iNOS, HSP70, Glut-1, and Glut-4 were upregulated in response to preconditioning. Significant elevation of HSP70 mRNA levels persisted up to 24 h after preconditioning. It is conceivable that upregulation of Glut-1, Glut-4, and iNOS is mediated by HIF because they are known HIF-1 target genes (19, 25, 32). Furthermore, expression of a constitutively stable hybrid HIF-1
protected cultured neonatal cardiomyocytes against simulated ischemia-reperfusion injury and induced a similar set of protective genes. These results suggest that HIF-1 is involved in the development of late-phase preconditioning in cultured neonatal cardiomyocytes by inducing multiple protective genes. It should be noted that the involvement of HIF in preconditioning of cultured cardiomyocytes against ischemia-reperfusion injury has been demonstrated by overexpression experiments only. The cause-effect relationship remains to be established by eliminating the expression of the endogenous
-subunits of HIF in cultured cardiomyocytes in vitro and/or myocardium in vivo.
Limitations of studies. In our studies, cultured neonatal cardiomyocytes were subjected to simulated ischemia-reperfusion (23). Adult cardiomyocytes die or dedifferentiate in long-term culture, which is required for manipulating gene expression and elucidating the molecular mechanisms underlying the late phase of preconditioning. Neonatal cardiomyocytes reasonably recapitulate the key features of ischemic preconditioning, including simulated ischemia, the temporal relationship between initiating and lethal simulated ischemia, the involvement of ligands to G protein-coupled receptors, and protein kinase C dependence (23). However, cultured neonatal cardiomyocytes are phenotypically different from working adult cardiomyocytes in the whole heart in vivo. In addition, these cells undergo isolation and culture manipulation and cannot be subjected to true ischemia-reperfusion. Because of these limitations, the molecular mechanisms observed in the cultured neonatal cardiomyocyte model may not reflect those in the aged working heart in vivo.
In summary, we have demonstrated that adenovirus-mediated expression of HIF-1/VP16 or HIF-1
/NF-
B, constitutively stable hybrid transcriptional factors, protects cultured neonatal cardiomyocytes against simulated ischemia-reperfusion injury by inducing multiple protective genes. Several lines of evidence suggest the involvement of HIF-1 in late-phase preconditioning in cultured neonatal cardiomyocytes. Our study provides new insights into the mechanism underlying the protection of cultured neonatal cardiomyocytes by simulated ischemic preconditioning.
![]() |
FOOTNOTES |
---|
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.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Arstall MA, Zhao YZ, Hornberger L, Kennedy SP, Buchholz RA, Osathanondh R, and Kelly RA. Human ventricular myocytes in vitro exhibit both early and delayed preconditioning responses to simulated ischemia. J Mol Cell Cardiol 30: 10191025, 1998.[CrossRef][ISI][Medline]
3. Barger PM and Kelly DP. PPAR signaling in the control of cardiac energy metabolism. Trends Cardiovasc Med 10: 238245, 2000.[CrossRef][ISI][Medline]
4. Baxter GF, Goma FM, and Yellon DM. Involvement of protein kinase C in the delayed cytoprotection following sublethal ischaemia in rabbit myocardium. Br J Pharmacol 115: 222224, 1995.[Abstract]
5. Belanger AJ, Lu H, Date T, Liu LX, Vincent KA, Akita GY, Cheng SH, Gregory RJ, and Jiang C. Hypoxia up-regulates expression of peroxisome proliferator-activated receptor angiopoietin-related gene (PGAR) in cardiomyocytes: role of hypoxia inducible factor 1
. J Mol Cell Cardiol 34: 765774, 2002.[CrossRef][ISI][Medline]
6. Bergeron M, Gidday JM, Yu AY, Semenza GL, Ferriero DM, and Sharp FR. Role of hypoxia-inducible factor-1 in hypoxia-induced ischemic tolerance in neonatal rat brain. Ann Neurol 48: 285296, 2000.[CrossRef][ISI][Medline]
7. Bergeron M, Yu AY, Solway KE, Semenza GL, and Sharp FR. Induction of hypoxia-inducible factor-1 (HIF-1) and its target genes following focal ischaemia in rat brain. Eur J Neurosci 11: 41594170, 1999.[CrossRef][ISI][Medline]
8. Bolli R. The late phase of preconditioning. Circ Res 87: 972983, 2000.
9. Bruick RK and McKnight SL. A conserved family of prolyl-4-hydroxylases that modify HIF. Science 294: 13371340, 2001.
10. Bunn HF and Poyton RO. Oxygen sensing and molecular adaptation to hypoxia. Physiol Rev 76: 839885, 1996.
11. Date T, Belanger AJ, Mochizuki S, Sullivan JA, Liu LX, Scaria A, Cheng SH, Gregory RJ, and Jiang C. Adenovirus-mediated expression of p35 prevents hypoxia/reoxygenation injury by reducing reactive oxygen species and caspase activity. Cardiovasc Res 55: 309319, 2002.[CrossRef][ISI][Medline]
12. Epstein AC, Gleadle JM, McNeill LA, Hewitson KS, O'Rourke J, Mole DR, Mukherji M, Metzen E, Wilson MI, Dhanda A, Tian YM, Masson N, Hamilton DL, Jaakkola P, Barstead R, Hodgkin J, Maxwell PH, Pugh CW, Schofield CJ, and Ratcliffe PJ. C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation. Cell 107: 4354, 2001.[ISI][Medline]
13. Gray MO, Karliner JS, and Mochly-Rosen D. A selective -protein kinase C antagonist inhibits protection of cardiac myocytes from hypoxia-induced cell death. J Biol Chem 272: 3094530951, 1997.
14. Grimm C, Wenzel A, Groszer M, Mayser H, Seeliger M, Samardzija M, Bauer C, Gassmann M, and Reme CE. HIF-1-induced erythropoietin in the hypoxic retina protects against light-induced retinal degeneration. Nat Med 8: 718724, 2002.[CrossRef][ISI][Medline]
15. Gross GJ. ATP-sensitive potassium channels and myocardial preconditioning. Basic Res Cardiol 90: 8588, 1995.[ISI][Medline]
16. Imagawa J, Baxter GF, and Yellon DM. Genistein, a tyrosine kinase inhibitor, blocks the "second window of protection" 48 h after ischemic preconditioning in the rabbit. J Mol Cell Cardiol 29: 18851893, 1997.[CrossRef][ISI][Medline]
17. Jayakumar J, Suzuki K, Khan M, Smolenski RT, Farrell A, Latif N, Raisky O, Abunasra H, Sammut IA, Murtuza B, Amrani M, and Yacoub MH. Gene therapy for myocardial protection: transfection of donor hearts with heat shock protein 70 gene protects cardiac function against ischemia-reperfusion injury. Circulation 102: III302III306, 2000.[Medline]
18. Jiang C, Lu H, Vincent KA, Shankara S, Belanger AJ, Cheng SH, Akita GY, Kelly RA, Goldberg MA, and Gregory RJ. Gene expression profiles in human cardiac cells subjected to hypoxia or expressing a hybrid form of HIF-1. Physiol Genomics 8: 2332, 2002.
19. Jung F, Palmer LA, Zhou N, and Johns RA. Hypoxic regulation of inducible nitric oxide synthase via hypoxia inducible factor-1 in cardiac myocytes. Circ Res 86: 319325, 2000.
20. Kawata H, Yoshida K, Kawamoto A, Kurioka H, Takase E, Sasaki Y, Hatanaka K, Kobayashi M, Ueyama T, Hashimoto T, and Dohi K. Ischemic preconditioning upregulates vascular endothelial growth factor mRNA expression and neovascularization via nuclear translocation of protein kinase C in the rat ischemic myocardium. Circ Res 88: 696704, 2001.
21. Kuzuya T, Hoshida S, Yamashita N, Fuji H, Oe H, Hori M, Kamada T, and Tada M. Delayed effects of sublethal ischemia on the acquisition of tolerance to ischemia. Circ Res 72: 12931299, 1993.[Abstract]
22. Li RC, Ping P, Zhang J, Wead WB, Cao X, Gao J, Zheng Y, Huang S, Han J, and Bolli R. PKC modulates NF-
B and AP-1 via mitogen-activated protein kinases in adult rabbit cardiomyocytes. Am J Physiol Heart Circ Physiol 279: H1679H1689, 2000.
23. Marber MS. Ischemic preconditioning in isolated cells. Circ Res 86: 926931, 2000.
24. Marber MS, Mestril R, Chi SH, Sayen MR, Yellon DM, and Dillmann WH. Overexpression of the rat inducible 70-kD heat stress protein in a transgenic mouse increases the resistance of the heart to ischemic injury. J Clin Invest 95: 14461456, 1995.[ISI][Medline]
25. Melillo G, Taylor LS, Brooks A, Musso T, Cox GW, and Varesio L. Functional requirement of the hypoxia-responsive element in the activation of the inducible nitric oxide synthase promoter by the iron chelator desferrioxamine. J Biol Chem 272: 1223612243, 1997.
26. Mestril R, Chi SH, Sayen MR, O'Reilly K, and Dillmann WH. Expression of inducible stress protein 70 in rat heart myogenic cells confers protection against simulated ischemia-induced injury. J Clin Invest 93: 759767, 1994.[ISI][Medline]
27. Murry CE, Jennings RB, and Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 74: 11241136, 1986.[Abstract]
28. Ovelgonne JH, Van Wijk R, Verkleij AJ, and Post JA. Cultured neonatal rat heart cells can be preconditioned by ischemia, but not by heat shock. The role of stress proteins. J Mol Cell Cardiol 28: 16171629, 1996.[CrossRef][ISI][Medline]
29. Plumier JC, Ross BM, Currie RW, Angelidis CE, Kazlaris H, Kollias G, and Pagoulatos GN. Transgenic mice expressing the human heat shock protein 70 have improved post-ischemic myocardial recovery. J Clin Invest 95: 18541860, 1995.[ISI][Medline]
30. Prass K, Ruscher K, Karsch M, Isaev N, Megow D, Priller J, Scharff A, Dirnagl U, and Meisel A. Desferrioxamine induces delayed tolerance against cerebral ischemia in vivo and in vitro. J Cereb Blood Flow Metab 22: 520525, 2002.[ISI][Medline]
31. Radford NB, Fina M, Benjamin IJ, Moreadith RW, Graves KH, Zhao P, Gavva S, Wiethoff A, Sherry AD, Malloy CR, and Williams RS. Cardioprotective effects of 70-kDa heat shock protein in transgenic mice. Proc Natl Acad Sci USA 93: 23392342, 1996.
32. Semenza GL. HIF-1, O2, and the 3 PHDs: how animal cells signal hypoxia to the nucleus. Cell 107: 13, 2001.[ISI][Medline]
33. Sugden PH and Clerk A. "Stress-responsive" mitogen-activated protein kinases (c-Jun N-terminal kinases and p38 mitogen-activated protein kinases) in the myocardium. Circ Res 83: 345352, 1998.
34. Vincent KA, Shyu KG, Luo Y, Magner M, Tio RA, Jiang C, Goldberg MA, Akita GY, Gregory RJ, and Isner JM. Angiogenesis is induced in a rabbit model of hindlimb ischemia by naked DNA encoding an HIF-1/VP16 hybrid transcription factor. Circulation 102: 22552261, 2000.
35. Webster KA, Discher DJ, and Bishopric NH. Cardioprotection in an in vitro model of hypoxic preconditioning. J Mol Cell Cardiol 27: 453458, 1995.[ISI][Medline]
36. Xuan YT, Tang XL, Banerjee S, Takano H, Li RC, Han H, Qiu Y, Li JJ, and Bolli R. Nuclear factor-B plays an essential role in the late phase of ischemic preconditioning in conscious rabbits. Circ Res 84: 10951109, 1999.
37. Yamakawa M, Liu LX, Date T, Belanger AJ, Vincent KA, Akita GY, Kuriyama T, Cheng SH, Gregory RJ, and Jiang C. Hypoxia-inducible factor-1 mediates activation of cultured vascular endothelial cells by inducing multiple angiogenic factors. Circ Res 93: 664673, 2003.
38. Zhao J, Renner O, Wightman L, Sugden PH, Stewart L, Miller AD, Latchman DS, and Marber MS. The expression of constitutively active isotypes of protein kinase C to investigate preconditioning. J Biol Chem 273: 2307223079, 1998.