A Novel Role of MicroRNA in Late Preconditioning
Upregulation of Endothelial Nitric Oxide Synthase and Heat Shock Protein 70
MicroRNAs (miRNAs) are noncoding RNAs of 18 to 24 nucleotides that are involved in posttranscriptional regulation of protein expression. Their role in ischemic preconditioning (IPC) is currently unknown. We hypothesized that miRNAs induced after IPC in the heart may create a preconditioned phenotype through upregulating proteins including endothelial nitric oxide synthase (eNOS)/inducible nitric oxide synthase (iNOS) and heat shock protein (HSP)70, which are implicated in the late-phase protection of IPC. miRNAs were extracted from hearts of ICR mice following IPC. The purified miRNAs were injected in vivo into the left ventricular wall of mice, and, 48 hours later, the hearts were subjected to regional ischemia/reperfusion injury by left anterior descending artery ligation for 30 minutes followed by reperfusion for 24 hour. IPC caused no changes in miRNA-23b and miRNA-483 whereas miRNA-1, miRNA-21and miRNA-24 were significantly increased. The IPC-miRNA treatment caused an increase in eNOS mRNA and protein, whereas iNOS was not changed. HSF-1 (heat shock transcription factor 1) and HSP70 were also increased with IPC-miRNA treatment versus control. Moreover, injection of IPC-miRNA protected the hearts against ischemia/reperfusion injury, as shown by a reduction of infarct size as compared with saline or non-IPC miRNA-treated control. We conclude that IPC-induced miRNAs trigger cardioprotection similar to the delayed phase of IPC, possibly through upregulating eNOS, HSP70, and the HSP70 transcription factor HSF-1.
Ischemic preconditioning (IPC) is a powerful cardioprotective phenomenon whereby repeated brief episodes of ischemia protect the heart against future myocardial infarction.1 Genetic reprogramming emerging during or following IPC, which simulates angina in the clinical setting can be characterized as protective in nature. Several mechanisms for IPC have been proposed, which broadly include the release of endogenous mediators including adenosine, activation of G-coupled receptors and protein kinase C, and synthesis of cytoprotective proteins including endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), cyclooxygenase-2, and heat shock protein (HSP)70, which either individually or in concert lead to protection against ischemia/reperfusion (I/R) injury.2,3
In recent years, microRNAs (miRNAs) have emerged as novel regulators of gene expression. The miRNAs are a family of small RNA with average length of 24 nucleotides that is too short to code for any proteins. For a long time, miRNAs were considered as byproducts of mRNA transcription, or even as “evolutionary transcriptional debris.” However, recent studies suggest that miRNAs participate in many cellular processes, such as apoptosis, fat metabolism, cell differentiation, tumorigenesis, and cardiogenesis.4 miRNAs are also critically involved in the pathological process of adult hearts, including cardiac hypertrophy,5 angiogenesis, arrhythmogenesis, and heart failure. We recently observed that whole body heat shock leads to synthesis of several miRNAs, which lead to protection against I/R injury.6 However, the potential role of endogenous miRNAs in IPC has never been investigated.
In the present study, we tested the hypothesis that miRNAs induced by IPC play an important role in protection against myocardial I/R injury. We induced miRNAs by short bursts of global ischemia and reperfusion in the isolated perfused hearts. The induced miRNAs (IPC-miRNAs) were then injected directly into the myocardium in vivo 48 hours before I/R injury. For the first time, our results show that miRNAs cause significant reduction in infarct size, which is associated with the upregulation of protective proteins including eNOS, HSF-1 (heat shock transcription factor 1), and HSP70 that are implicated in the delayed phase of IPC in the heart.
Materials and Methods
Details of the IPC/infarction protocol, miRNA isolation, their verification, and injection, as well as measurement of infarct size are provided in the online data supplement at http://circres.ahajournals.org.
Results and Discussion
In the Langendorff model, IPC stimulus (2 bursts of 30 seconds global ischemia followed by 90 seconds reperfusion; Figure 1A) reduced infarct size from 29.7±2.1% in control group to 9.1±1.8% in the IPC group (69.3% reduction, mean±SEM, P<0.05; Figure 1B). The IPC protocol caused no changes in miRNA-23b and miRNA-483 (Figure 1C) but caused a significant induction of miRNA-1 (162±13%), miRNA-21 (118±6%), and miRNA-24 (46±12%) as compared to control (Figure 1D). To determine the cause–effect relationship between IPC-induced endogenous miRNAs and cardioprotection, we injected the pool of extracted miRNAs from non-IPC and IPC hearts directly into the left ventricular wall in situ in a separate set of mice (miRNA-injected group). Forty-eight hours later, the mice were subjected to I/R injury in vivo by ligation of left coronary artery for 30 minutes followed by reperfusion for 24 hours. Our results show that miRNAs derived from IPC hearts produced a protective phenotype with significantly lower infarction (18.8±2.5%) as compared to saline-injected controls (37.5±2.2%) or miRNAs prepared from non-IPC hearts (39.3±2.3%). There was no difference in infarct size between saline-injected controls versus non-IPC miRNA-treated hearts. Also, there were no significant differences in risk areas between the groups (Figure 2B). To confirm the delivery of miRNA by intramyocardial injection, RT-PCR was performed and the results illustrate a significant uptake of miRNA-21 1 hour after injection in the risk zone (Figure 3A).
To gain further insight into the mechanisms underlying miRNA-induced protection, we probed several target protective proteins that are implicated in IPC, including eNOS, iNOS, HSP70, and the HSP70 transcription factor HSF-1. As shown in Figure 3A, induction of eNOS mRNA (61±6.7%) was detected in the IPC-miRNA group 2 hours following treatment. However, no changes in iNOS mRNA were observed. Western blot analysis confirmed a significant upregulation in eNOS protein (62.0±6.7%) and HSF-1 (42.7±3.0%) 4 hours after IPC-miRNA treatment. HSP70 was also significantly increased (102.3±8.9%) 48 hours after IPC-miRNA treatment. Again, similar to mRNA, iNOS protein was not significantly changed (Figure 3C and 3D).
Despite potential species differences in cardioprotection,7 it is widely known that the protective effects of IPC occur in an early phase that develops rapidly after the initial stimulus but dissipates within 2 to 3 hours and a late phase that becomes apparent 12 to 24 hours later and persists for ≈72 hours. The role of NO-derived from eNOS in the late phase of IPC has been suggested previously.8 Although the critical role of iNOS has also been well documented in the late phase of IPC9 and pharmacological preconditioning,10,11 we did not observe upregulation of this protein in the IPC-miRNA–treated hearts. This is possibly attributable to the strength of the IPC stimulus, which may not be sufficiently potent to trigger miRNA changes for synthesis of iNOS. A previous study has shown that 6 cycles of 4-minute coronary occlusion/4-minute reperfusion cause significant increase in myocardial iNOS.9 In this study, we used a less stringent IPC protocol, ie, 2 bursts of 30 seconds ischemia and 90 seconds reperfusion.
The induction of HSPs by stressful stimuli such as elevated temperature or ischemia12 is mediated by the HSP70 transcription factor HSF-1. It is known that the cytoplasmic HSF-1 monomer forms a trimer and moves to the nucleus, where it binds to its target sites (known as heat shock elements) in the regulatory regions of the HSP genes. Following its phosphorylation, HSF-1 induces expression of HSP70 that could protect hearts against ischemic injuries during the late phase of IPC. Our results show that IPC-miRNAs can induce HSF-1 and HSP70, which may play a role in ischemic tolerance observed in the heart. However, we do not know which particular miRNA is responsible for the increased synthesis of HSF-1/HSP70 or eNOS in the present study. miRNA-1 has been linked in posttranscriptional repression of HSP60 and HSP70 in H9c2 cells which is in contrast to the upregulation of HSP70 synthesis observed in the present study. Also, eNOS introns contain miRNAs which regulate eNOS expression.13 Although we observed a significant upregulation of miRNA-1, miRNA-21, and miRNA-24, it is possible that other miRNAs may have caused increased expression of the cytoprotective proteins as well. It is known that miRNAs generally function as inhibitory mechanisms of gene expression, and, therefore, it is possible that suppression of genes participating in injurious processes during I/R injury may underlie miRNA-induced protection in the heart. At the same time, it is possible that the suppression of injurious genes may lead to upregulation of protective proteins including eNOS and HSP70, as shown in the present study. Identification of such injurious genes is critical for validation of this hypothesis. Nevertheless, the protection observed against I/R injury in this study clearly suggests that concerted action of one or perhaps several miRNAs, induced following IPC, may have been responsible for the increased expression of eNOS and HSP70. Further studies are needed to identify the miRNA(s) from IPC that could increase the cytoprotective proteins without adverse side effects. The delivery of such miRNA(s) in the heart would have immense therapeutic potential in reducing myocardial infarction in patients with heart disease.
Sources of Funding
This study is supported by NIH grants HL51045, HL59469, HL79424, and HL093685 to (R.C.K.).
↵*Both authors contributed equally to this work.
Original received December 22, 2008; revision received February 3, 2009; accepted February 3, 2009.
Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation. 1986; 74: 1124–1136.
Bolli R. The late phase of preconditioning. Circ Res. 2000; 87: 972–983.
Heusch G, Boengler K, Schulz R. Cardioprotection: nitric oxide, protein kinases, and mitochondria. Circulation. 2008; 118: 1915–1919.
Sayed D, Hong C, Chen IY, Lypowy J, Abdellatif M. MicroRNAs play an essential role in the development of cardiac hypertrophy. Circ Res. 2007; 100: 416–424.
Skyschally A, van Caster P, Boengler K, Gres P, Musiolik J, Schilawa D, Schulz R, Heusch G. Ischemic postconditioning in pigs: no causal role for RISK activation. Circ Res. 2009; 104: 15–18.
Xuan YT, Tang XL, Qiu Y, Banerjee S, Takano H, Han H, Bolli R. Biphasic response of cardiac NO synthase isoforms to ischemic preconditioning in conscious rabbits. Am J Physiol Heart Circ Physiol. 2000; 279: H2360–H2371.
Guo Y, Jones WK, Xuan YT, Tang XL, Bao W, Wu WJ, Han H, Laubach VE, Ping P, Yang Z, Qiu Y, Bolli R. The late phase of ischemic preconditioning is abrogated by targeted disruption of the inducible NO synthase gene. Proc Natl Acad Sci U S A. 1999; 96: 11507–11512.
Xi L, Jarret N, Hess ML, Kukreja RC. Essential role of inducible nitric oxide in monophosphoryl lipid A induced delayed ischemic protection: evidence from pharmacological inhibition and gene knockout mice. Circulation. 1999; 99: 2157–2163.
Salloum F, Yin C, Xi L, Kukreja RC. Sildenafil (Viagra) induces delayed preconditioning through iNOS-dependent pathway in mouse heart. Circ Res. 2003; 92: 595–597.
Nishizawa J, Nakai A, Matsuda K, Komeda M, Ban T, Nagata K. Reactive oxygen species play an important role in the activation of heat shock factor 1 in ischemic-reperfused heart. Circulation. 1999; 99: 934–941.
Zhang MX, Ou H, Shen YH, Wang J, Wang J, Coselli J, Wang XL. Regulation of endothelial nitric oxide synthase by small RNA. Proc Natl Acad Sci U S A. 2005; 102: 16967–16972.