In recent years, tremendous effort has been made in utilizing a miR microarray screening approach to address the miR expression profiling in infarcted hearts from animal models and human patients.14–20
However, the role of a specific miR in ischemic heart disease is just emerging. In the present study, we center on elucidating the functional consequences of miR-494 in vivo
, using gain-of-function and loss-of-function approaches. While miR-494 has been reported to be downregulated in the hypertrophic heart,18
chronic overexpression of miR-494 in vivo
did not yield any cardiac morphological or pathological abnormalities ( and Supplemental Fig. S4
). More excitingly, we observed that miR-494-overexpressing hearts were resistant to I/R-induced injury in vivo
and ex vivo
; whereas knockdown of endogenous miR-494 by antagomiR showed the opposite effect. Taken together, these data clearly demonstrate that increased levels of miR-494 render cardioprotection against I/R injury. Our study also suggests that down-regulation of miR-494 observed in human patients14
may be causally involved in the progression of heart failure, at least in part.
A prominent characteristic of miR-494 is that it potentially regulates both anti-apoptotic (e.g. CDK6, FGFR2, IGF2BP1, XIAP, HLF, LIF, API5, FGF7, IGF1R, HDGF, etc.) and pro-apoptotic (e.g. SOCS6, PTEN, ROCK1, PPARGC1α, CAMKIIδ, etc) proteins. Therefore, it is plausible that miR-494 may function as either pro-apoptotic or anti-apoptotic depending on the cellular environment and cell-specific expressed targets. For instance, in JHU-021 cancer cells, transfection of miR-494 induced cell death, indicating its pro-apoptotic role, 11
although the underlying mechanisms remain unknown. In cardiomyocytes, our results have implicated miR-494 as an anti-apoptotic microRNA via activation of the Akt signaling pathway. While we did verify that three pro-apoptotic proteins (PTEN, ROCK1, CAMKIIδ) and two anti-apoptotic proteins (FGFR2 and LIF) were bona fide
targets of miR-494, the individual contribution of these targets to the downstream Akt pathway may be unequal. In miR-494-overexpressing cardiomyocytes, the impact of PTEN, ROCK1, and CAMKIIδ (either individually or in combination) on the Akt signaling may be more significant than those of FGFR2, LIF, or other anti-apoptotic targets, thereby tilting the balance of the Akt pathway towards activation (). These interesting findings suggest that divergent targets of a miR may work unequally to balance a common signaling pathway and eventually affect its functional consequences.
In addition, we performed mRNA array analysis for miR-494 TG hearts and observed that a total of 685 mRNAs were significantly altered (upregulated: 292; downregulated: 393, supplemental Tables S1 and S2
). Surprisingly, among these 685 mRNAs, only 6 are predicted to be targets of miR-494 by TargetScan. More intriguingly, mRNA levels of PTEN, ROCK1, CAMKIIδ, FGFR2 and LIF showed no alteration (supplemental Tables S3
), while their protein levels were downregulated in miR-494-cardiomyocytes. The inconsistency between mRNA and protein levels suggests that the chronic miR-overexpressing model may have limitations when using conventional mRNA expression arrays to detect miR targets. Another possibility may be that miR-494 dampens target-protein levels mostly by impeding their translation, rather than by breaking down their messenger RNAs. Notably, a recent study using human bronchial epithelial cells33
showed that miR-494 regulated PTEN protein expression, but not mRNA levels, which is consistent with our present findings. To further identify the whole target network for miR-494, a new technique named SILAC (stable isotope labeling with amino acids in cell culture) 34, 35
will be an ideal tool; however, such analysis may fall outside the scope and intent of this study.
Currently, it is well accepted that inhibition of PTEN, ROCKs, or CaMKII has a major impact on improving myocardial survival following an I/R episode.26–29
While several pharmacological agents can effectively inhibit PTEN, ROCKs or CaMKII, these inhibitors may be toxic in physiological settings and their clinical applications may be restricted.36, 37
Thus, it would be very important to identify novel substitutes that orchestrate suppression of PTEN, ROCKs and CaMKII in the myocardium. In the present study, we clearly showed that overexpression of miR-494 reduced the levels of PTEN, ROCK1 and CaMKIIδ, which might work in concert to activate the Akt signaling, a critical survival pathway in the myocardium. However, it may be experimentally complicated to link PTEN, ROCK1 and CaMKIIδ together for elucidating mechanisms underlying the protective effects of miR-494 against I/R. Further studies are needed to ascertain the causal relationship between miR-494-modulated targets and their functional consequences.
In conclusion, to our best knowledge, this is the first report to demonstrate the protective role of miR-494 in I/R is mediated, at least in part, via activation of the Akt-mitochondrial signaling. The present observations expand our understanding of miRs associated with ischemic heart disease and may provide a basis for novel therapeutic strategies aimed at enhancing cardiomyocyte survival in the ischemic myocardium.
MicroRNAs (miRs), a new class of non-protein-coding small RNAs, have emerged as regulators that control the expression of hundreds of proteins. As a consequence, they may widely influence the signaling networks leading to pathological or physiological responses such as myocardial ischemia/reperfusion-induced injury and ischemia preconditioning-elicited cardioprotection. While miR expression has been profiled in infarcted hearts from animal models and human patients, the role of a specific miR in ischemic heart disease is just emerging. Uncovering miRs as important regulators not only for single genes but also for whole gene networks has enormous therapeutic implications. In the present study, we discovered that increased levels of mature miR-494 rendered cardioprotection against ischemia/reperfusion (I/R)-induced injury, whereas knockdown of endogenous miR-494 by administration of antagomiR-494 sensitized hearts to I/R injury. Importantly, we identified that miR-494 targeted PTEN, ROCK1 and CAMKIIδ in cardiomyocytes, consequently, activated the Akt signaling pathway. These data suggest that down-regulation of miR-494 observed in human failing hearts may be causally involved in the progression of heart failure, at least in part. Therefore, systemically or locally administration of miR-494 may introduce the newest prospect for the management of ischemic heart disease.