Several studies have shown that heat shock treatment protects the heart against ischemia/reperfusion injury [33
]. The specific mechanisms underlying heat shock protection include synthesis of heat shock proteins [34
], antioxidant defenses [35
], enhanced mitochondrial respiration [36
]. In addition, it has been shown that heat shock protects by opening of mitochondrial KATP
] and causes resistance to opening of mitochondrial permeability transition pore [38
], which may contribute to heat shock protection against cellular injury through inhibition of apoptosis. In the present study, we have observed a significant induction of miRNA-1, miRNA-21 and miRNA-24 following whole body heat shock in the heart. Moreover, mice treated with miRNAs isolated from the heat shocked mice demonstrated significantly reduced infarct size in the heart following global ischemia and reperfusion. Similarly, injection of chemically synthesized exogenous miRNA-21 reduced infarct size and the co-treatment with the 2′-O-methyl miRNA – which blocks miRNA-21 through antisense inhibition abolished the protective effect. Except for Bnip3, miRNA injection caused downregulation of pro-apoptotic proteins including caspases 1, 2, 8 and 14, Bid, Bcl10, Cidea, Ltbr, Trp53 and Fasl, while anti-apoptotic proteins including Bag3, and Prdx2 were increased. These results suggest a potential role of miRNAs in reducing myocardial infarction through repression of apoptotic genes and upregulation of anti-apoptotic proteins.
Although only three miRNA, namely miRNA 1, 21 and 24, were verified in the present study, heat shock may well induce many other miRNAs. We did not perform experiments to demonstrate whether these intraperitoneally injected miRNAs ended up in the heart. Nevertheless, a recent study showed that the simple systemic delivery of a unconjugated locked-nucleic-acid-modified oligonucleotide (LNA-antimiR) effectively antagonized the liver-expressed miRNA-122 [39
]. Acute administration by intravenous injections of LNA-antimiR in monkeys resulted in uptake of the LNA-antimiR in the cytoplasm of primate hepatocytes and formation of stable heteroduplexes between the antimiR and miRNA-122. This was accompanied by depletion of mature miRNA-122 and dose-dependent lowering of plasma cholesterol. Our data also supports these findings because the chemically synthesized miRNA-21 reduced infarct size in the heart which was blocked with miRNA-21 inhibitor. These data suggest the possibility that the physiological effect of the miRNA21 and its antagonist were actually occurring in the heart following intraperitoneal injection.
Apoptosis is a major cause for cardiac infarction following ischemia-reperfusion [40
]. miRNA-1 is preferentially expressed in cardiac muscle [42
] and has been shown to regulate apoptosis. The inhibition of miRNA-21 has been shown to suppress cell growth by increasing apoptosis and decreasing cell proliferation [43
]. In contrast, knockdown of miR-21 in cultured glioblastoma cells triggers activation of caspases and leads to increased apoptotic cell death [7
]. miRNA-24 has recently been shown to be involved in the inhibition of skeletal muscle differentiation by TGF-β which provides clues for mechanisms underlying the physiological roles of the growth factor during myogenesis [44
]. The attenuation of myocardial infarction with miRNA in the present study may be related to reduced expression of apoptotic genes, Bid and Bcl-10 which may account for the observed protection since increased Bid and Bcl-10 can bind to Bcl-2 to promote apoptosis. On the other hand, Bag-3 may compete with Bid and Bcl-10 to bind to Bcl-2 to reduce apoptosis [45
]. miRNA induction may also reduce infarct size through additional cellular processes other than apoptosis. For example, the increased levels of Prx2 observed in the current study may protect heart against oxidative stress since Prx2 is an extremely efficient scavenger of hydrogen peroxide [46
In the present study, Bnip3 was increased in the heart following miRNA treatment. Although it is well recognized to be an apoptotic gene, some studies suggest that BNIP3 is not sufficient for cell death but rather plays a critical role in hypoxia-induced autophagy [47
], Moreover, it has been suggested that rather than promoting death, BNIP3 may actually allow survival either by preventing ATP depletion or by eliminating damaged mitochondria. [47
]. Such a function of BNIP3 may be subverted under conditions associated with acidosis that arise following extended periods of hypoxia and anaerobic glycolysis. Bnip3 is also shown to be expressed in healthy adult heart without evidence of cell death [48
]. This finding is in line with the study by Tracy et. al., who found that Bnip3 allowed cells to survive by preventing ATP depletion or by eliminating damaged mitochondria [47
]. Overexpression of Bnip3 in HL-1 cardiac myocytes subjected to simulated ischemia /reperfusion, caused upregulation of autophagic activity which constituted a protective response against Bnip3 mediated death signaling [49
In conclusion, for the first time, we have provided evidence for the potential role of endogenously synthesized miRNA’s in cardioprotection following ischemia/ reperfusion injury. These miRNA have many advantages over other exogenous agents. For example, they are natural cellular products and therefore, non toxic to cells. They can be induced in vivo under natural conditions, such as hyperthermia. Due to their short length, miRNAs can also easy to move around and cross sub-cellular structures. Therefore, identifying the role of endogenously synthesized miRNAs in protective pathophysiological stimuli including ischemic, heat shock and by pharmacological preconditioning means may open up novel strategies to protect the heart in patients with coronary artery disease.