Microarray analyses indicate that ischemic and pharmacological preconditioning suppress overexpression of the non-long terminal repeat retrotransposon long interspersed nuclear element 1 (LINE-1, L1) after ischemia-reperfusion in the rat heart. We tested whether L1 overexpression is mechanistically involved in postischemic myocardial damage. Isolated, perfused rat hearts were treated with antisense or scrambled oligonucleotides (ODNs) against L1 for 60 min and exposed to 40 min of ischemia followed by 60 min of reperfusion. Functional recovery and infarct size were measured. Effective nuclear uptake was determined by FITC-labeled ODNs, and downregulation of L1 transcription was confirmed by RT-PCR. Immunoblot analysis was used to assess changes in expression levels of the L1-encoded proteins ORF1p and ORF2p. Immunohistochemistry was performed to localize ORF1/2 proteins in cardiac tissue. Effects of ODNs on prosurvival protein kinase B (Akt/PKB) expression and activity were also determined. Antisense ODNs against L1 prevented L1 burst after ischemia-reperfusion. Inhibition of L1 increased Akt/PKBβ expression, enhanced phosphorylation of PKB at serine 473, and markedly improved postischemic functional recovery and decreased infarct size. Antisense ODN-mediated protection was abolished by LY-294002, confirming the involvement of the Akt/PKB survival pathway. ORF1p and ORF2p were found to be expressed in rat heart. ORF1p showed a predominantly nuclear localization in cardiomyocytes, whereas ORF2p was exclusively present in endothelial cells. ORF1p levels increased in response to ischemia, which was reversed by antisense ODN treatment. No significant changes in ORF2p were noted. Our results demonstrate that L1 suppression favorably affects postischemic outcome in the heart. Modifying transcriptional activity of L1 may represent a novel anti-ischemic therapeutic strategy.
- long interspersed nuclear element 1 retrotransposons
brief episodes of sublethal cardiac ischemia produce a marked protection against subsequent prolonged potentially lethal ischemia (35). This phenomenon is termed ischemic preconditioning and is one of the most powerful means of attaining myocardial protection. Similarly, preconditioning can be elicited and amplified by pharmacological agents. During the past two decades, research in the field of cardiac preconditioning has identified a number of signaling steps equally involved in ischemic and pharmacological preconditioning (53). These comprise activation of G protein-coupled receptors, protein kinase C, and sarcolemmal and mitochondrial ATP-sensitive potassium (KATP) channels as well as formation of reactive oxygen and nitrogen species (34). Using microarray analysis, we recently compared (5) the transcriptional responses of ischemic preconditioning and pharmacological preconditioning by the volatile anesthetic isoflurane, two highly protective strategies against ischemia-reperfusion injury. This survey revealed a cluster of transcripts related to long interspersed nuclear element 1 (LINE-1, L1) retrotransposons being consistently upregulated after ischemia-reperfusion (Fig. 1A).
L1 is a mammalian autonomous non-long terminal repeat retrotransposon, and L1 copies cover ∼17–25% of the genome (27). A functional full-length L1 consists of a 5′-untranslated region (UTR) with an internal promoter, two open reading frames (ORFs), and a 3′-UTR containing the poly(A) tail (Fig. 1B). ORF1 encodes a ∼40-to 46-kDa nucleic acid binding protein with chaperone activity (ORF1p; Refs. 30–32), and ORF2 encodes a ∼150-kDa bifunctional polypeptide with both reverse transcriptase and DNA endonuclease activity (ORF2p; Ref. 49). The presence of both functional proteins is an essential prerequisite for L1 retrotransposition. To date, 377 full-length L1 have been annotated in the rat genome (39), but numerous truncated, rearranged, or mutated L1 insertions still retain the ability to be expressed. Retrotransposons have been found to induce genomic alterations by retrotranspositional insertion or homologous recombination (6, 18, 38). L1 activity has been linked to gene disruption (38), transcriptional regulation (13), amplification of nonautonomous retrotransposons such as pseudogenes and short interspersed nuclear element (SINE) family members (50), and most recently cell growth and differentiation (7, 41). Although a growing body of evidence points to an important role of L1 retrotransposition in the evolution of the mammalian genome, little is known about the biological function of L1 in terminally differentiated tissues.
Intriguingly, expression of L1 transcripts was associated with decreased function and increased infarct size, whereas inhibition of L1 expression by ischemic and pharmacological preconditioning closely paralleled improved postischemic outcome (5). Furthermore, we were able to show (26) that isoflurane administered in the reperfusion phase also prevented the induction of L1 transcription. These results prompted us to test whether enhanced L1 activity after ischemia-reperfusion merely reflects a cellular stress response or whether changes in L1 expression are mechanistically involved in ischemia-reperfusion damage of the heart. Specifically, we hypothesized that inhibition of L1 expression would decrease postischemic myocardial injury.
The results of our experiments provide evidence, for the first time, that L1-encoded ORF1p and ORF2p are expressed in adult rat cardiac tissue. Using an antisense oligonucleotide (AS-ODN) approach, we were able to demonstrate that inhibition of L1 expression during ischemia-reperfusion favorably affects functional and structural outcome in the heart, which appears to be in part mediated by prosurvival Akt/PKB signaling.
MATERIALS AND METHODS
Experimental protocols used in this investigation were approved by the Animal Care and Use Committee of the University of Zurich. All procedures conformed to the “Guiding Principles in the Care and Use of Animals” of the American Physiological Society and were in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Pub. No. 85-23, revised 1996).
Isolated, perfused rat heart preparation.
Male Wistar rats (300 g) were heparinized (500 U ip) and decapitated 20 min later. The hearts were removed and perfused in a Langendorff apparatus with Krebs-Henseleit buffer (mM: 155 Na+, 5.6 K+, 138 Cl−, 2.1 Ca2+, 1.2 PO43−, 25 HCO3−, 0.56 Mg2+, 11 glucose) gassed with 95% O2-5% CO2 (pH 7.4, 37°C). A water-filled balloon was inserted into the left ventricle and inflated to set an end-diastolic pressure of 0–5 mmHg during the initial equilibration. Perfusion pressure was set to 80 mmHg. Perfusion pressure, epicardial ECG, coronary flow, and contractility [positive and negative change of pressure with time (±dP/dt)] were measured as previously described (48).
Spontaneously beating hearts were equilibrated for 10 min and subsequently exposed to AS-ODN or scrambled oligonucleotides (SCRAM) in a recirculating mode for 1 h. Pretreated and untreated hearts were subjected to 40 min of normothermic no-flow ischemia and reperfused for 1 h. The control group consisted of time-matched perfused hearts (170 min of perfusion). In some experiments, 15 μM 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one hydrochloride (LY-294002; Alexis, Lausen, Switzerland), a specific inhibitor of phosphatidylinositol 3-kinase (PI3-kinase), was administered for 15 min immediately at the onset of reperfusion (47). LY-294002 was dissolved in DMSO at a final concentration of <0.1%. For each experimental group, five hearts were prepared and functional parameters were recorded (Fig. 2). An additional five hearts of each group were used to determine infarct size by 1% triphenyltetrazolium chloride staining, as previously described (26).
L1 antisense and scrambled sequences of ODNs.
Phosphorothioated ODNs were obtained from AlphaDNA (Montreal, QC, Canada). The sequences of the L1Rn-specific 20-mer AS-ODN (GenBank accession no. M13100) were 5′-AGTTTCTGGCGAGAAGTCTG-3′ (ODN1), 5′-CGATAGCACGCATGGGATTA-3′ (ODN2), and 5′-CTGTACCAATCACCATGCAG-3′ (ODN3; see Fig. 1B). The DNA sequence of the scrambled oligos (SCRAM) was 5′-ATTAGGGTACGCACGATAGC-3′. A cocktail of the three antisense sequences, each at a concentration of 200 nM, was used in the antisense experiments (total concentration of 600 nM). SCRAM was administered at a concentration of 600 nM. In some experiments, 5′-end fluorescein-labeled ODNs were used to confirm successful cellular penetration and nuclear uptake.
At the end of the experiments, left ventricular tissue was frozen in liquid nitrogen and stored at −80°C. Hearts were powdered in liquid nitrogen and homogenized in TRIzol LS reagent (Invitrogen, Basel, Switzerland) and chloroform-isoamyl alcohol (Fluka, Buchs, Switzerland). The aqueous phase was mixed with isopropanol and precipitated overnight at −20°C. The pellet was washed with isopropanol, dried at 37°C, and eluted in diethyl pyrocarbonate-treated H2O. RT-PCR was performed to detect transcription of L1, SINEs, Akt1/PKBα, and Akt2/PKBβ. The primers were synthesized by Microsynth (Balgach, Switzerland) and are listed in Table 1. For each amplification, 20 μl of cDNA was diluted in water (1:10) before being used as template for the QuantiTect Sybr Green RT-PCR kit (Qiagen, Hilden, Germany). RT-PCR quantification and determination of expression levels were performed on the ABI Prism 7700 Sequence Detector real-time PCR machine (Perkin-Elmer, Foster City, CA). Amplification reactions were conducted with an initial step at 90°C for 3 min followed by 20–35 cycles. All PCR reactions were performed in triplicate, and α-tubulin and methionine aminopeptidase 2 were used as controls. The expected size of PCR products was confirmed by agarose gel electrophoresis.
Generation of monoclonal antibodies directed against L1Rn ORF1p and ORF2p.
To generate monoclonal antibodies specifically recognizing ORF1p, an ORF1-specific PCR product covering nt 1068–1844 of L1 cDNA rL1-21 (accession no. DQ100480) was inserted into the multiple cloning site of the bacterial expression vector pET.32b (Merck Biosciences, Bad Soden, Germany), leading to pET32b-ORF1del1–143. Similarly, an ORF2-specific PCR product ranging from nt 2642 to nt 3204 of L1 cDNA rL1-8 (accession number DQ100475) was cloned into the pET32b vector, giving rise to the expression vector pET32b-ORF2M292–480. The constructs were used to express peptides encompassing residues 144–402 of ORF1p (accession no. DQ100480) and residues 292–480 of ORF2p (accession no. DQ100475), respectively, in frame with an NH2-terminal thioredoxin (Trx) and 6x histidine (6xHis) tag in Escherichia coli. Trx-6xHis-ORF1pdel1–143 and -ORF2pM292–480 were purified from soluble protein fractions and inclusion bodies via the cobalt IMAC resin Talon (BD Biosciences, Heidelberg, Germany) technique. Trx-6xHis-ORF1pdel1–143 and -ORF2pM292–480 fusion proteins were purified by electroelution from preparative SDS-PAGE gels and used for the immunization of BALB/c mice. Immunization and preparation of hybridoma cells was carried out as described in standard protocols (2). Primary analysis and screening of hybridoma cells was performed as published by Hawkes (15). The same antigens as for immunization were used, employing the immunodot-blotting method with native proteins immobilized on a nitrocellulose membrane (0.45 μm; Schleicher & Schuell, Dassel, Germany) fixed in a 96-well dot blot chamber (Minifold, Schleicher & Schuell). Hybridoma clones rG24 (isotype IgG2) and 2H9 (isotype IgM) were obtained from mice that were immunized with Trx-6xHis-ORF1pdel1–143 and -ORF2pM292–480 fusion proteins. The specificity of the monoclonal antibodies was further tested by immunofluorescence staining of transfected cells, immunoprecipitation, and immunoblotting (Kirilyjuk A, Schumann GG, Tolstonog GV, Buschmann C, Grikscheit K, and Traub P, unpublished observation).
Rat chloroleukemia (RCL) cells were obtained from the Paul-Ehrlich Institute. Polyclonal anti-PKB antibody (Ab10) was a gift from Dr. Brian Hemmings, Friedrich Miescher Institute (Basel, Switzerland) (9). Polyclonal antibody specific for phospho-PKB (serine 473) was obtained from Cell Signaling Technology (Beverly, MA). Monoclonal anti-actin was purchased from Chemicon (Temecula, CA) and anti-histone H1 from Santa Cruz Biotechnology (Santa Cruz, CA). Left ventricular tissue was powdered and homogenized in lysis buffer containing (mM) 50 Tris·HCl at pH 7.5, 120 NaCl, 10 NaF, 40 β-glycerol phosphate, 0.1 sodium orthopervanadate, 1 phenylmethylsulfonyl fluoride, 1 microcystin-LR (Alexis, Lausen, Switzerland), and 1% Triton X-100. Extracts were centrifuged for 30 min at 12,000 g, and protein concentrations in the supernatant were determined by the Bradford method. Rat heart powder was swollen for 5 min on ice in solvent A (mM: 50 Tris·HCl pH 7.5, 1 dithiothreitol, 2 EDTA, 2 EGTA, and 1 benzamidine, with 0.2 μM phenylmethylsulfonyl fluoride) with 300 mM sucrose and extracted on ice with a Polytron homogenizer. The homogenate was centrifuged at 1,000 g for 10 min to separate crude nuclear pellets from the cytoplasmic fraction. The crude nuclear pellet was quickly washed twice with solvent A, exposed for 30 min to Triton X-100 lysis buffer on ice, and centrifuged at 12,000 g for 30 min. The resulting supernatant served as nuclear fraction and was used for ORF1p immunoblotting. For all other blots, total tissue extracts were used. Extracts were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Amersham Biosciences, Little Chalfont, UK). Membranes were blocked with PBS containing 5% nonfat dry milk and 0.05% Tween 20 for 1 h and then incubated with the primary antibody. The membranes were washed and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Pierce, Rockford, IL). After extensive washing, the blots were exposed to films (Fuji Photo Film, Kanakawa, Japan) for various times to obtain a linear response by the enhanced chemiluminescence method (Pierce, Rockford, IL). The quantity of the immunoreactive bands was determined by densitometry (MCID Imaging, Fonthill, ON, Canada).
Immunohistochemistry of L1-encoded ORF1p and ORF2p.
Subcellular localization of ORF1p and ORF2p was determined by immunofluorescence staining. At the end of the procedure, hearts were perfused with 5 μM propidium iodide for 5 min to allow detection of necrotic cells. Left ventricular tissue samples were placed in optimal cutting temperature (OCT) medium (Tissue-Tek, Sakura Finetek, Torrance, CA), frozen in liquid nitrogen, and stored at −80°C. Cryosections (7 μm) were prepared with a cryostat (Cryo-star HM 560M, Microtom, Kalamazoo, MI) and collected on gelatin-precoated slides. All sections were fixed for 10 min in 0.5% paraformaldehyde at room temperature, rinsed with PBS, and incubated in 10% normal goat serum for 30 min to block nonspecific binding. Sections were incubated for 90 min at room temperature, using the same antibodies as for Western blotting. Polyclonal platelet endothelial cell adhesion molecule 1 (PECAM-1) antibody (Santa Cruz Biotechnology) was used to stain the endothelium, and polyclonal myosin binding protein C antibody, a kind gift from Dr. Jean-Claude Perriard (Institute of Cell Biology, Swiss Federal Institute of Technology, Zurich, Switzerland), was used to selectively stain cardiomyocytes (1). Sections were washed with PBS and incubated for 1 h with a mixture of secondary antibodies conjugated to Alexa Fluor 555 rabbit anti-mouse or Alexa Fluor 488 goat anti-rabbit (Molecular Probes/Invitrogen, Basel, Switzerland) (1:500). 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI; Sigma, St. Louis, MO; 10 ng/ml in PBS) was used as a marker for nuclei. Sections were protected with coverslips and DAKO mounting medium (DAKO, Carpinteria, CA) and analyzed by epifluorescence microscopy with an upright microscope (Axioplan2, Zeiss, Jena, Germany), with appropriate filter blocks for the detection of FITC, Cy3, and UV fluorescence.
Repeated-measures analysis of variance was used to evaluate differences over time between groups. Unpaired t-tests were used to compare groups at identical time points and paired t-tests to compare within groups over time (SigmaStat version 2.0; SPSS Science, Chicago, IL). Post hoc Tukey tests for multiple comparisons was used. P < 0.05 was considered to be statistically significant. Data are presented as means(SD).
L1-specific AS-ODN prevent L1 expression burst after ischemia-reperfusion in isolated, perfused rat hearts.
To test whether AS-ODN successfully penetrated cell membranes and accumulated in the nuclei of myocardial cells, hearts were perfused with FITC-labeled L1-specific ODN or FITC alone for 1 h followed by 10 min of washout. With fluorescence microscopy, paraformaldehyde-fixed and DAPI-stained cryosections were used to determine successful nuclear ODN uptake. Although FITC alone did not accumulate in nuclei of myocardial cells, FITC-labeled ODNs clearly colocalized with the nuclear marker DAPI (Fig. 3A). RT-PCR was applied to confirm that AS-ODN effectively and specifically led to a reduction of L1 mRNA after ischemia-reperfusion. mRNA levels of both L1s (Fig. 3B) and SINEs (Fig. 3C) were markedly increased after 40 min of ischemia and 60 min of reperfusion. The burst of L1 expression was selectively abolished when AS-ODN were added 60 min before test ischemia. Conversely, no change in L1 transcript levels was observed when SCRAM were used. Importantly, although SINE (ID_Rn) exhibited some decline after antisense treatment against L1, both SINEs transcripts remained elevated (Fig. 3C). These results clearly confirm the effectiveness and selectivity of the antisense treatment used.
Inhibition of L1 transcriptional burst improves postischemic functional recovery and decreases infarct size.
In separate experiments, hearts (n = 5 per experimental group) were used to explore the effect of AS-ODN treatment on postischemic functional recovery (Table 2) and structural damage (Fig. 4). Infarct size and functional recovery were determined after 40 min of test ischemia and 60 min of reperfusion. Antisense treatment decreased infarct size from 35%(SD2) in untreated hearts to 9%(SD4). In contrast, the administration of the PI3-kinase inhibitor LY-294002 during early reperfusion completely abolished the AS-ODN-mediated protection against ischemia-reperfusion injury [infarct size 33%(SD5); Fig. 4]. No change in infarct size was observed after administration of SCRAM. In parallel to decreased infarct size, markedly improved functional recovery was observed in AS-ODN-treated hearts (Table 2). These results suggest that activation of the PI3-kinase-Akt/PKB signaling pathway is involved in the observed improved functional recovery and decreased structural damage in AS-ODN-treated hearts exposed to ischemia-reperfusion. To further corroborate our findings, we determined the effect of the inhibition of L1 expression on prosurvival Akt/PKB expression and phosphorylation. Although antisense treatment significantly increased Akt2/PKBβ transcriptional levels (Fig. 5A), no change in total Akt/PKB protein level was detected 1 h after ischemia (Fig. 5B). In contrast, phosphorylation of Akt/PKB at serine 473, a marker for Akt/PKB activation, was enhanced in AS-ODN-treated hearts compared with untreated hearts or hearts exposed to SCRAM (Fig. 5B).
L1-encoded ORF1p and ORF2p are expressed in adult rat hearts and are differentially regulated in response to ischemia-reperfusion.
Using monoclonal antibodies against ORF1p and ORF2p (Kirilyjuk A, Schumann GG, Tolstonog GV, Buschmann C, Grikscheit K, and Traub P, unpublished observation), we tested whether the L1 transcripts would also be translated in myocardial tissue. Expression of ORF1p and ORF2p was measured 1 h after reperfusion in the different treatment protocols. Immunohistochemical analysis revealed both cytoplasmic and nuclear localization of ORF1p in cardiomyocytes (Fig. 6, A and B). ORF1p was clearly detected in the nuclear fraction of cardiac tissue and was significantly increased in hearts exposed to ischemia-reperfusion compared with control hearts (time-matched perfusion) (Fig. 6C). Treatment with AS-ODN but not SCRAM reduced ORF1p expression to control levels. Interestingly, ORF2p was exclusively detected in endothelial cells of ischemic as well as control tissues, where it clearly colocalized with the endothelial cell marker PECAM-1 (Fig. 7, A and B). In contrast to ORF1p, ORF2p did not show significant changes after ischemia-reperfusion (Fig. 7C). These results show for the first time that L1-encoded proteins are expressed in adult cardiac tissue and that ORF1p levels are increased after ischemia-reperfusion.
Our study investigating a possible involvement of L1 expression in postischemic myocardial damage led to the following salient findings. First, inhibition of L1 expression during ischemia-reperfusion improves postischemic functional recovery, decreases infarct size, and enhances prosurvival Akt/PKB signaling. These results indicate that peri-ischemic L1 activity is not merely a response of the heart to cellular stress but rather induces an injury-promoting effect in ischemia-reperfusion. Peri-ischemic downregulation of L1 expression activated Akt/PKB, one of the most powerful prosurvival signaling pathways in the heart (33). Second, we were able to demonstrate that the L1-encoded ORF1 and ORF2 proteins are expressed in the rat heart. ORF1p but not ORF2p expression was significantly upregulated in response to ischemia-reperfusion. Notably, this is the first time that L1 gene products were detected in the adult mammalian heart. Although ORF1p has been reported to be localized only or predominantly in the cytoplasm to date (3, 29, 46), direct and indirect evidence is accumulating that ORF1p is also localized in the nucleus, at least in certain stages of the cell cycle in specific cell types (12, 29). In addition, the nucleic acid chaperone activity of ORF1p suggests a role in mediating nucleic acid transfer steps during L1 reverse transcription (31), implying the localization of ORF1p in tight association with nuclear genomic DNA. Consistent with our observations, the presence of ORF2p in vascular endothelial cells and its apparent association with various stages of cellular differentiation were recently reported (7). However, the physiological function of L1-encoded proteins—apart from their role in transposition—remains elusive. Together, our experiments point to an as yet unknown biological effect of L1 expression in the mammalian heart.
The importance of Akt/PKB in mediating cell survival and cytoprotection in the context of ischemic and pharmacological pre- and postconditioning has been well established (4, 14, 45, 47). Key substrates of PKB include glycogen synthase kinase-3β and FOXOs, a subgroup of the Forkhead family of transcription factors, both of which are involved in the regulation of cell growth and apoptosis. In particular, glycogen synthase kinase-3β has been functionally related to cytoprotection under conditions of elevated formation of reactive oxygen species (20). In our experiments, we noted a transcriptional upregulation of the inducible isoform Akt2/PKBβ in response to AS-ODN treatment against L1 expression, but no changes in total Akt/PKB protein levels were detected. Conversely, phosphorylation of Akt/PKB at serine 473 was increased by ∼30% after 60 min of reperfusion in hearts subjected to AS-ODN-mediated downregulation of L1 expression (Fig. 5B). Importantly, Akt/PKB activation was accompanied by a significant reduction in infarct size, and this protection was abolished by the PI3-kinase inhibitor LY-294002 (Fig. 4). Although the precise events underlying this phenomenon remain elusive and require additional investigation, some potential molecular mechanisms surrounding the observed effects have recently emerged from research in the fields of retrotransposable elements and epigenetics and should be briefly discussed.
Stress-induced transcriptional burst of retrotransposable elements.
Ischemia-reperfusion induced an increase in expression of both L1s and SINEs (Fig. 3). The measured increase in SINE RNA observed in this study, together with our previously reported (5) transient expression of heat shock protein mRNAs in similar experiments, confirm earlier observations from other laboratories (21, 25, 40) and further support the concept that SINEs might be considered “stress genes.” Replication and transposition of SINEs, however, requires a concomitant cellular reverse transcriptase activity, which was shown to be provided by the L1 protein machinery (50). Elevated amounts of L1 transcripts were also found in patients with immune-mediated diseases, another paradigm of cellular stress. Neidhart et al. (36) demonstrated that L1-related transcripts are expressed in synovial tissue samples of patients with rheumatoid arthritis. In synovial fibroblasts, it was also shown that ORF1 induction correlated with increased levels of p38δ MAPK in an ORF2-independent manner (23). These results and our own observations support the concept that changes in L1 transcription and/or its translational products may epigenetically regulate the expression of key cellular kinases and thereby affect signal transduction.
How is L1 expression linked to ischemia-reperfusion injury?
The activation of L1 elements may be a consequence of chromatin opening and/or activation of genes involved in the cardiac response to ischemia. Histone acetylation, which relaxes chromatin structure and promotes transcription, may be promoter dependent and may play an important role in the cellular response to stress (17, 44). Paradigmatically, the work by Illi and colleagues (17) underscores the role of MAPKs in controlling the response to stress by histone modification. Activated MAPKs—a hallmark of ischemia-reperfusion-induced signal transduction—phosphorylate transcription factors and regulators of transcription (16), thereby altering gene expression. p38 MAPKs favor histone acetylation and the induction of early genes such as c-fos.
Once activated, L1 elements in close proximity to important genes may have a significant impact on their level of expression by inhibition of transcriptional elongation or by changes in promoter activity (13, 42, 51). These observations offer a possible explanation of why controlling L1 transcription is cardioprotective. A search among L1 elements in the rat genome annotated in the L1Base database (39) revealed several potentially relevant genes containing L1 sequences either within their introns or in the close genomic neighborhood. Candidates with documented roles in cardioprotection are catalase (11) and insulin 1 (10, 19). Catalase, an important antioxidant enzyme involved in cellular protection against oxidative stress, is transcriptionally regulated by FOXOs (37), which, in turn, are targets of PKB. Insulin administered to the heart at the onset of reperfusion protects the heart from ischemia-reperfusion injury in a PKB-mediated way (10, 19, 52). Interestingly, the L1 repetitive sequence found close to the promoter region of the rat insulin gene was shown to repress its transcription (24). Therefore, one may postulate that active L1 sequences may interfere with cardiac survival programs induced by ischemia-reperfusion.
AS-ODN against L1 expression as potential therapeutic strategy.
In this study, ex vivo low-pressure-mediated application of phosphorothioated ODNs via the coronary bed under normothermic, quasi-physiological conditions achieved nuclear localization of fluorescent (FITC)-labeled L1-specific ODNs and markedly decreased L1 transcription. Methods of ODN delivery under hypothermic high-pressure conditions were previously reported to be successful in downregulating target gene expression in the heart (28). It is believed that this form of application triggers pathways circumventing endocytosis with lysosomal degradation of ODNs (43). We found that our modified normothermic ODN delivery was rapidly effective, similar to earlier in vivo studies (8), and, most importantly, devoid of cytotoxic complications as observed with lipid formulations or viral particles. Therefore, peri-ischemic inhibition of L1 by this method might represent a novel anti-ischemic therapeutic strategy.
Limitations of study.
Detailed analyses will be required to understand the complex temporal relationship between the development of myocardial infarction and activation of L1 and to better delineate the regulatory functions of L1 RNA and L1-encoded proteins in ischemia-reperfusion damage of the heart. Furthermore, future studies should help us to understand the mechanisms by which AS-ODN against L1 lead to sustained phosphorylation of Akt/PKB. Although the observed increase in Akt/PKB phosphorylation was relatively small (after 60 min of reperfusion), a significant reduction in infarct size was achieved. This implies that additional protective mechanisms may be involved, which should be investigated in the future.
In conclusion, our results demonstrate for the first time that both L1-encoded proteins are expressed in adult rat cardiac tissue. Using an AS-ODN approach, we were able to show that inhibition of L1 expression during ischemia-reperfusion favorably affects functional and structural outcome in the rat heart, which appears to be in part mediated by Akt/PKB signaling.
This work was supported by Grant No. 3200B0-103980/1 of the Swiss National Science Foundation, Berne, Switzerland; the Swiss University Conference, Berne, Switzerland; a grant of the Swiss Heart Foundation, Berne, Switzerland; a grant from Abbott Switzerland, Baar, Switzerland; a grant from Novartis Foundation, Basel, Switzerland; a grant from the Olga Mayenfisch Foundation, Zurich, Switzerland; a grant from the Swiss Society of Anaesthesiology and Reanimation, Berne, Switzerland; and a grant from the International Anesthesia Research Society, Cleveland, OH (to M. Zaugg). This work was also supported in part by Grants SCHU 1014/5-1 and 1014/5-2 from the Deutsche Forschungsgemeinschaft (to G. G. Schumann).
Present address of R. da Silva: Hemodynamics and Cardiovascular Technology Laboratory, Swiss Federal Institute of Technology, Lausanne, Switzerland.
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).
Address for reprint requests and other correspondence: M. Zaugg and E. Lucchinetti, Cardiovascular Anesthesia Research Laboratory, Institute of Anesthesiology, E-HOF, University Hospital Zurich, Rämistrasse 100, CH-8091 Zurich, Switzerland (E-mails:, )
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