Abstract 377: Endogenously Induced Micro-RNAs by Ischemic Preconditioning Trigger Cardioprotection Through Upregulation of eNOS and HSP70
Background: miRNAs are a class of non-coding RNAs of 18–24 nucleotides (nt) that post-transcriptionally regulate protein expression. We recently showed that endogenously induced miRNAs by ischemic preconditioning (IPC) play important role in protection of the heart against ischemia/reperfusion (I/R) injury. However, the target proteins involved in the protective effect of miRNAs remain unknown. We hypothesized that miRNAs may create preconditioned phenotype through up-regulating proteins including eNOS/iNOS and HSP70 that have been implicated in the protective effect of IPC.
Methods and Results: miRNAs were extracted from hearts of ICR mice (n=6) following the IPC protocol (2 cycles of 30 sec of normothermic global ischemia) in Langendorff mode. The purified miRNAs were injected in vivo into the left ventricle wall of mice (n=6). Real time PCR and Western blot were performed on hearts injected with either miRNA extracted from IPC hearts (IPC-miRNA group), or miRNA extracted from non-treated hearts (control). To confirm the role of IPC-miRNA in cardioprotection, a subset of mice were treated with miRNA and subjected to I/R injury in vivo by left coronary artery ligation for 30 min followed by reperfusion for 24 hr. Our results show significant increase in eNOS mRNA (+61 ± 6.7%, mean±SEM) in the IPC-miRNA group after 2-hour treatment. Also, there was 92.0 ± 8.1% increase in eNOS protein and 42.7 ± 3.0% increase in heat shock transcription factor 1 (HSF-1) after 4-hours of treatment. HSP-70 was also increased after 48-hour treatment (+102.3 ± 8.9% in IPC-miRNA group versus control). Interestingly, iNOS, the mediator of delayed phase of IPC was not significantly altered by miRNA treatment. The miRNA injected hearts reproduced preconditioning phenotype (as demonstrated by significant reduction of infarct size as compared to saline-treated control). This protection was abolished in the hearts treated with specific inhibitors of miRNA-1 and miRNA-21.
Conclusion: Endogenous miRNAs induced by IPC trigger cardioprotection, possibly through up-regulating protective proteins including eNOS, HSP70 and its transcription factor HSF-1.