In the Langendorff model, IPC stimulus (2 bursts of 30 sec global ischemia followed by 90 sec reperfusion, ) 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, ). The IPC protocol caused no changes in miRNA-23b and miRNA-483 (), but caused a significant induction of miRNA-1 (162±13%), miRNA-21 (118 ± 6%), and miRNA-24 (46 ± 12%) as compared to control (). To determine the cause-and-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). As shown in , a significant uptake of miRNA-21 was evident after 1 h of injection in the risk zone. Forty-eight h later, the mice were subjected to I/R injury in vivo by ligation of left coronary artery for 30 min followed by reperfusion for 24 h. 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 ().
Induction of miRNA by ischemic preconditioning (IPC) in the Langendorff isolated perfused heart
Effect of miRNA on induction of cardioprotective proteins
Effect of miRNA on myocardial infarct size following ischemia/reperfusion
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, HSP 70 and its transcription factor, HSF-1. As shown in , induction of eNOS mRNA (61±6.7%) was detected in the IPC-miRNA group 2 h following treatment. However, no changes in iNOS mRNA were observed. Western blot analysis confirmed a significant up-regulation in eNOS protein (62.0±6.7%) and HSF-1 (42.7±3.0%) 4 h after IPC-miRNA treatment. HSP70 was also significantly increased (102.3±8.9%) 48 h after IPC-miRNA treatment. Again, similar to mRNA, iNOS protein was not significantly changed ().
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 2–3 h, and a late phase that becomes apparent 12–24 h later and persists for approximately 72 h. 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 IPC (9
) and pharmacological preconditioning (10
), we did not observe upregulation of this protein in the IPC-miRNA-treated hearts. This is possibly due 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 six cycles of 4-min coronary occlusion/4-min reperfusion cause significant increase in myocardial iNOS (9
). In this study, we utilized a less stringent IPC protocol i.e., 2 bursts of 30 sec ischemia and 90 sec reperfusion.
The induction of heat shock proteins by stressful stimuli such as elevated temperature or ischemia (12
) is mediated by heat shock 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 or direct gene transfer of protective proteins as reported previously. Our results show that IPC-miRNAs can induce HSF-1 and HSP 70 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 post-transcriptional 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 HSP 70 as shown in the current 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.