Micro RNA-411 Expression Improves Cardiac Phenotype Following Myocardial Infarction in Mice

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SUMMARY
Induction of endogenous regenerative capacity has emerged as one promising approach to repair damaged hearts following myocardial infarction (MI). Re-expression of factors that are exclusively expressed during embryonic development may reactivate the ability of adult cardiomyocytes to regenerate. Here, we identified miR-411 as a potent inducer of cardiomyocyte proliferation. Overexpression of miR-411 in the heart significantly increased cardiomyocyte proliferation and survival in a model MI. We found that miR-411 enhances the activity of YAP, the main downstream effector of the Hippo pathway, in cardiomyocytes. In   Post-MI adverse remodeling is, at least in part, attributable to the limited capacity of adult cardiomyocytes to regenerate. 2 However, recent advances in the understanding of cardiomyocyte cell cycle control have indicated that reactivating endogenous regenerative capacity in cardiomyocytes may become a promising approach to repair the infarcted heart. 3,4 It is understood that cardiac growth during embryonic development mainly occurs through proliferation of cardiomyocytes (hyperplasia). 5,6 In contrast, postnatal heart growth predominantly occurs via cardiomyocyte enlargement (hypertrophy) because terminally differentiated cardiomyocytes normally become mitotically inactive. 7 Therefore, it is not surprising that the pattern of global gene expression in embryonic hearts is significantly different from that of postnatal hearts. 8  including cardiovascular diseases. 9 In the heart, several miRNAs have been demonstrated to modulate cardiac hypertrophy/heart failure, 10,11 fibrosis, 12 and cardiac pacemaker function. 13,14 More interestingly, several studies have identified miRNAs that can induce cardiomyocyte proliferation. [15][16][17] The possibility of inhibiting miRNA function using antagomirs or enhancing their biological activity using miRNA mimics makes miRNA an attractive therapeutic approach for cardiovascular diseases, including adverse heart remodeling and heart failure post-MI.
Based on this knowledge, we reasoned that miR-  types. For example, in liver, bone, and lung cancer cells, miR-411 promotes cell proliferation, [19][20][21][22] whereas in ovarian, breast, bladder, and cervical cancers, miR-411 inhibits cell proliferation. 23 NRCMs, and its expression was significantly higher compared to that in NRCFs ( Figure 1A). In contrast, we did not observe miR-411 expression in CVECs. We then analyzed the level of miR-411 expression in neonatal and adult rat hearts. miRNA-enriched RNA was isolated from 3-day-old rat neonate hearts and fully grown adult rat hearts (12 weeks old). We found that miR-411 expression was significantly higher in neonate hearts compared to adult rat hearts ( Figure 1B).
( Figure 2A). Interestingly, the level of miR-411 at the distant sites was similar to that of control hearts ( Figure 2B). This suggests that up-regulation of miR-411 appeared to be spatially confined to the injection site at day 5 postinjection.
To follow-up these findings, we examined the expression of YAP target genes in mouse heart tissues after intramyocardial injection with miR-411. As shown in Figure 6G, we detected a significant increase in Ctgf expression and a trend toward elevated Ankrd1 and Birc5 at the injection sites, whereas at distant sites, we did not observe any elevations of YAP-target gene expression ( Figure 6H).
We went on by analyzing the YAP subcellular location using a GFP-YAP construct. As shown in Figures 7A and 7B Because miRNAs are known to exert their biological effects primarily by reducing the expression of their target genes, 9 we examined the expression level of the core components of the Hippo pathway. Surprisingly, we did not observe any changes in the expression of Hippo core kinases in NRCMs after transfection with miR-411 mimics ( Figures 7E to 7L).
However, we observed a significant reduction in LATS1 phosphorylation following miR-411 overexpression ( Figures 6M and 6N), which was in agreement with the finding that YAP phosphorylation was reduced. Together, these data indicate that in cardiomyocytes, miR-411 modulates the Hippo kinase cascade without altering their total protein level, suggesting that the direct target of miR-411 might be outside of the Hippo core kinases, with subsequent effects on LATS1 and/or YAP activity.  (B to E) one-way analysis of variance followed by post hoc test for multiple pairwise comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, Nugroho et al

DISCUSSION
The main finding of this study is that re-expression of a factor (ie, miR-411) that normally is expressed dur- Another important factor that may contribute to the reduced infarct size is the reduction of apoptosis.  [35][36][37] The antiapoptotic property of miR-411 might be, at least in part, caused by its ability to induce YAP activity. Increased YAP activity is associated with increased expression of antiapoptosis and prosurvival genes such as connective tissue growth factor (CTGF), 38 cysteine-rich angiogenic inducer 61 (CYR61), 39 and survivin. 40 We also found that miR-411 reduced LATS1 phosphorylation. Because LATS1 is known as a strong proapoptotic kinase, 41 this may contribute to the protective effect of miR-411 treatment.
Surprisingly, we did not observe any changes in the expression of the core components of the Hippo  hours postinjection until 12 days postinjection. 16,47,48 In this study, we demonstrated that nanopolymer PEI can successfully deliver miRNA mimics into the heart,

CONCLUSIONS
In conclusion, our study shows that overexpression of miR-411 in the heart can improve cardiac remodeling and function following MI. This will add to a growing body of evidence suggesting that miRNA treatments may provide a promising approach to control adverse cardiac remodeling post-MI. 15,17, 48 We also found that the regenerative effect of miR-411 might involve the modulation of the Hippo pathway. This is in agreement with recent data showing that many regenerative miRNAs are associated with the Hippo pathway. 17,49 FUNDING SUPPORT AND AUTHOR DISCLOSURES

PERSPECTIVES COMPETENCY IN MEDICAL KNOWLEDGE:
Following an acute MI, the heart undergoes adverse cardiac remodeling, encompassing processes including hypertrophy, dilatation, and fibrosis. These changes are at least in part caused by the inability of adult cardiomyocytes to regenerate following an infarction. It is hypothesized that re-expression of factors that are highly expressed during heart development may overcome this problem. We discovered that overexpression of miR-411 in cardiomyocytes induced cell proliferation and increased survival under stress. In an animal model of MI, we observed that treatment with miR-411 ameliorated adverse remodeling and improved heart function.
TRANSLATIONAL OUTLOOK: This study identified miR-411 as a candidate factor that can be targeted to control adverse heart remodeling following MI. As miRNA-based therapies have entered clinical trials for other diseases, it has been indicated that targeting of miR-411 is therapeutically feasible. Further studies using large animal models to confirm the therapeutic effects, to assess the long-term side effects/off-target effects, and to develop an effective delivery strategy will be needed to advance miR-411 toward translational application.
Nugroho et al