In this study, we reveal a novel role for miR-24 in the regulation of mammalian cardiomyocyte apoptosis. We found that miR-24 inhibits apoptosis in cardiomyocytes in vitro and in vivo using a mouse MI model. In addition, we identified Bim as one of the direct targets of miR-24 and, through epistasis analyses, found that Bim mediates a part of miR-24’s effect on apoptosis. Finally, introduction of miR-24 in vivo did not disturb the normal function of the heart but partially protected the heart from injury. The beneficial effects of miR-24 in MI hearts, while statistically significant by our measurement, are quite small and may be below the true sensitivity of the techniques being used. Future experimentation using more robust and accurate assays with a large sample number is needed for validation of applying this miRNA in treating ischemic cardiac disease. In addition, the improvement in heart function and reduction in scar size by overexpressing miR-24 might be caused by other functions of miR-24 independent of apoptosis or a combination of all. Further cardiac function analyses and scar size measurements when inhibiting the antiapoptotic function of miR-24 (i.e., knocking down the downstream components in miR-24–mediated apoptosis pathway) will hopefully provide some clues.
Like most miRNAs, miR-24 functions in many other biological processes and pathways. miR-24 was first reported to negatively regulate erythroid differentiation through inhibition of human activin type I receptor ALK4 (Wang et al., 2008
). In addition, miR-24 appears to affect the tumor suppressor p16 (Lal et al., 2008
), the DNA repair process, and cell cycle regulation (Lal et al., 2009a
; Rogler et al., 2009
). Particularly relevant to this study, miR-24 is required to prevent apoptosis during normal development of the retina in frogs (Walker and Harland, 2009
), suggesting evolutionary conservation of miR-24 function, although the mechanisms may be divergent. These findings support the idea that a single miRNA, like miR-24, can regulate multiple independent pathways that may converge on a common biological outcome such as regulation of apoptosis. Other miRNAs, such as miR-21, may also play a role in regulating the cardiac response to ischemic injury (Thum et al., 2008
; Dong et al., 2009
miR-24 is expressed in both cardiomyocytes and fibroblasts. Our experiments demonstrate that miR-24 can exert antiapoptotic effects in cardiomyocytes in a cell-autonomous fashion. We and others have shown that cardiac fibroblasts can function as major signaling centers to affect the neighboring cardiomyocytes (Ieda et al., 2009
; Takeda et al., 2010
), and it is quite possible that miR-24 also functions in cardiac fibroblasts to promote cardiomyocyte survival. In this case, miR-24 would likely regulate pathways leading to paracrine secretion of survival factors that impinge on neighboring myocytes. Future studies of fibroblast-specific expression of miR-24 could help resolve this issue.
It is curious that an miRNA that represses an apoptotic factor such as Bim in the setting of ischemia would be down-regulated. One might expect that such an miRNA would be up-regulated to protect the cells from ischemic damage, particularly in the BZ which is attempting to survive in hypoxic conditions. However, the down-regulation may reflect the need to remove cells that are under ischemic stress. It will be interesting to determine whether the ischemia-induced miR-24 down-regulation has other more beneficial consequences through yet unknown targets.
The finding that miR-24 targets the Bcl2 family member Bim for repression highlights an important facet of miRNA-mediated regulation of critical cellular events. The dosage of Bim is apparently very important, as cells have engineered numerous mechanisms to quantitatively regulate its protein levels. Bim mediates both mitochondrial and ER stress–induced apoptosis and is regulated through multiple cellular modalities, including phosphorylation, ubiquitination, and proteosome-mediated degradation (Dijkers et al., 2000
; Puthalakath and Strasser, 2002
; Ley et al., 2005
; Puthalakath et al., 2007
). Our findings reveal yet another layer of posttranscriptional Bim regulation involving miR-24 and suggest that the cell uses numerous back-up mechanisms and regulatory pathways to carefully titrate the dose of this central regulator of apoptosis.