miRNAs and cardiomyocyte apoptosis
It is noticed that the previous work on miRNAs and apoptosis has been mostly limited to the context of cancer, while studies on apoptosis regulation by miRNAs in non-cancer cells have been sparse. The first evidence for the role of miRNAs in cardiomyocyte apoptosis was obtained in 2007 from my laboratory demonstrating the proapoptotic effect of miR-1 and anti-apoptotic effect of miR-133 in response to oxidative stress. Subsequent studies in 2010 and 2008 revealed the involvement of other miRNAs such as miR-21, miR-24 and miR-29 in ischemic myocardial injury[119,120]. The currently known apoptosis-regulating miRNAs in cardiomyocytes are illustrated in Figure 5.
Figure 5 Diagram illustrating the role and signaling mechanisms of miRNAs in apoptosis of cardiac cells.
Solid arrow indicates induction; dashed line indicates inhibition; and pause sign indicates repression. HG: High glucose; AMI: Acute myocardial infarction; I/R-I: Ischemia/reperfusion injury; ROS: Reactive oxygen species.
Role of miR-1 and miR-133: Oxidative stress constitutes one of the major threats to the living system and accumulative oxidative damage has been implicated in many degenerative conditions including the aforementioned diseases such as heart failure, atherosclerosis and Alzheimer’s disease[121-125], and even aging process, all of which are associated with progressive decrease in cell density. Indeed, oxidative stress is one of the most crucial factors causing neurodegenerative disorders. There is also a growing body of evidence that oxidative stress increases in myocardial failure and may contribute to the structural and functional changes that lead to disease progression. Moreover, oxidative stress is a well-known factor promoting apoptosis[126-129].
We found that miR-1 level is significantly increased in response to oxidative stress in cardiomyocytes. This overexpression is involved in regulation of apoptotic cell death. Overexpression of miR-1 in H9c2 rat myoblasts provoked apoptotic cell death, which was partially rescued by treatment with miR-133. Similar effects on oxidative stress-induced apoptosis were observed in hydrogen peroxide (H2O2)-treated H9c2 cells, wherein cotransfection with miR-1 led to 60% reduction in the IC50 value necessary for oxidative stress-induced DNA fragmentation, and cotransfection with miR-133 resulted in a 40% increase in the IC50 value required for oxidative stress-induced DNA fragmentation. Qualitatively similar results were obtained when rat neonatal ventricular myocytes were exposed to H2O2. Subsequent computational analyses predicted that heat shock protein HSP60 and HSP70 were targets for miR-1 and that caspase-9 was a target for miR-133. This was verified experimentally by showing that miR-1 suppressed HSP60 and HSP70 protein levels by about 70% and 60%, respectively, whereas miR-133 decreases protein levels of caspase-9 and caspase-9 activity levels in rat H9c2 cells. The post-transcriptional repression of HSP60 and HSP70 and caspase-9 was confirmed by luciferase reporter experiments. We postulated that the relative levels of miR-1 and miR-133 may determine cell fate.
It is noteworthy that expression of miR-1 is upregulated in coronary disease, acute myocardium ischemia (AMI) and ischemia/reperfusion injury (I/R-I)[85,130], but is downregulated in cardiac hypertrophy/heart failure[121,131,132], wherein apoptosis is an important mode of cell death[86,87]. In this regard, it seems that increase in miR-1 during AMI and I/R-I contributes to apoptosis in these settings, and decrease in miR-1 in cardiac hypertrophy/heart failure is an adaptive change to minimize its deleterious effect. On the other hand, the cytoprotective effect of miR-133 may be weakened in cardiac hypertrophy/heart failure since miR-133 level has been found significantly reduced under such conditions and this may contribute to increased tendency of apoptosis induction in hypertrophic myocytes[82-84]. These notes merits future studies to verify.
The proapoptotic action of miR-1 in cardiomyocytes has been subsequently reproduced by other laboratories. A 2008 study by Yu et al investigated the possible miRNA mechanism for glucose cardiotoxicity. Glucose toxicity is an important initiator of cardiovascular disease, contributing to the development of cardiomyocyte death and diabetic complications. Yu et al showed that H9C2 cells exposed to high glucose have increased miR-1 expression level, decreased mitochondrial membrane potential, increased cytochrome-c release, and increased apoptosis. Glucose induced mitochondrial dysfunction, cytochrome-c release and apoptosis were blocked by IGF-1. miR-1 mimics, but not mutant miR-1, blocked the capacity of IGF-1 to prevent glucose-induced mitochondrial dysfunction, cytochrome-c release and apoptosis. The finding indicates that IGF-1 inhibits glucose-induced mitochondrial dysfunction, cytochrome-c release and apoptosis via downregulating miR-1 expression. The finding, according to the authors, provides a miRNA mechanism for the deleterious effects of glucose in the development of cardiomyocyte death and diabetic complications.
In a most recent study in 2009, Tang et al studied the role of miR-1 in a rat model of I/R-I. The level of miR-1 was found inversely correlated with Bcl-2 protein expression in cardiomyocytes with I/R-I. The in vitro level of miR-1 was dramatically increased in response to H2O2. Overexpression of miR-1 facilitated H2O2-induced apoptosis in cardiomyocytes. Inhibition of miR-1 by antisense inhibitory oligonucleotides caused marked resistance to H2O2. Through bioinformatics, the authors identified the potential target sites for miR-1 on the 3’UTR of Bcl-2. miR-1 significantly reduced the expression of Bcl-2 at both mRNA and protein levels. The post-transcriptional repression of Bcl-2 was further confirmed by luciferase reporter experiments. The data indicate that miR-1 is not only proarrhythmic in AMI but also proapoptptic in I/R-I. The findings from these above studies also indicate that the proapoptotic action of miR-1 involves multiple death signaling pathways including HSP60/70, IGF-1 and Bcl-1, in a coordinated manner or separately under different situations.
Role of miR-21: In addition to miR-133, it appears that miR-21 is also an anti-apoptotic miRNA. miR-21 has been found upregulated in various cancers (malignant glioblastoma, colorectal carcinoma, cervical adenocarcinoma) and, based on its in silico-predicted proapoptotic gene targets, has been proposed as a potential antiapoptotic miRNA[71,114,115]. Knockdown of miR-21 in glioblastoma cells leads to caspase activation and apoptotic cell death, and depletion of miR-21 in vascular smooth muscle cells (VSMCs) results in a dose-dependent increase in apoptosis and decrease in cell proliferation. As noted above, several independent groups have shown that the expression levels of miR-21 are increased by 2-4 folds in hypertrophied heart[134-137]. Although miR-21 levels were not examined in relation to myocyte apoptosis in these studies, the upregulation of miR-21 may represent a prosurvival stress response in response to hemodynamic pressure overload.
Yin et al investigated the role of miRNA in protection against I/R-I in heart. Mice subjected to cytoprotective heat-shock (HS) showed a significant increase of miR-1 by 80%, miR-21 by 100% and miR-24 by 60% in the heart, as determined by qPCR. miRNAs isolated from HS mice and injected into non-HS mice significantly reduced infarct size after I/R-I, which was associated with the inhibition of pro-apoptotic genes and increase in anti-apoptotic genes. Chemically synthesized miR-21 also reduced infarct size, whereas a miR-21 inhibitor abolished this effect. These studies provide evidence for the potential role of endogenous miRNA in cardioprotection following I/R-I. miRNA treatment caused profound changes in several apoptotic related genes as determined by gene microarray analysis. The caspase family members 1, 2, 8 and 14 were suppressed in the hearts of HS mice treated with miRNA as compared with the controls. Except for BNIP-3, most of the pro-apoptotic genes, including Bid (BH3 interacting domain death agonist), Bcl-10 (B-cell leukemia/lymphoma 10), Cidea (cell death-inducing DNA fragmentation factor, alpha subunit-like effector A), Ltbr (lymphotoxin B receptor), Trp53 (transformation related protein 53), Fas (TNF receptor superfamily member) and Fasl (Fas ligand, TNF superfamily, member 6), were also repressed. On the other hand, the anti-apoptotic genes, Bag-3 (Bcl-2-associated athanogene and Prdx2 (Peroxiredoxin 2) were increased. According to previous studies, miR-1 has apoptosis-promoting effect[81,130,133], miR-21 is anti-apoptotic in cardiac cells, and miR-24 is anti-apoptotic in cancer cells. In particular, miR-1 was found to induce cardiomyocytes apoptosis in a rat model of I/R-I. It is possible that the anti-apoptotic effect of these miRNAs in I/R-I is a net outcome of the counteracting effects of these miRNAs. Moreover, most of the apoptosis-related proteins reported in this study are predicted not to be the targets for these miRNAs and whether the observed changes in their expression were ascribed to the upregulation of these miRNAs are not certain; by logic, the upregulation of the anti-apoptotic proteins could not be the direct consequence of the upregulation of the miRNAs.
In a subsequent study, Cheng et al confirmed the anti-apoptoptic effect of miR-21 in cardiac cells. Using quantitative real-time RT-PCR (qRT-PCR), the authors demonstrated that miR-21 was upregulated in cardiac myocytes after treatment with H2O2. H2O2-induced cardiac cell death and apoptosis were increased by miR-21 inhibitor and was decreased by pre-miR-21. PDCD4 was established as a direct target of miR-21 in cardiac myocytes. Pre-miR-21-mediated protective effect on cardiac myocyte injury was inhibited in H2O2-treated cardiac cells via adenovirus-mediated overexpression of PDCD4 without miR-21 binding site. Moreover, activator protein 1 was a downstream signaling molecule of PDCD4 that was involved in miR-21-mediated effect on cardiac myocytes. The results suggest that miR-21 is sensitive to H2O2 stimulation. miR-21 participates in H2O2-mediated gene regulation and functional modulation in cardiac myocytes. miR-21 might play an essential role in heart diseases related to oxidative stress such as cardiac hypertrophy, heart failure, myocardial infarction, and myocardial I/R-I.
Role of miR-29: Pioglitazone, a peroxisome proliferator-activated receptor (PPAR)-γ agonist, has been documented by numerous studies to be able to limit myocardial infarct size in experimental animals[115,116]. Ye et al assessed the effects of PPAR-γ activation on myocardial miRNA expression and the role of miRNAs in I/R-I in the rat heart after pioglitazone administration using miRNA microarray methods, followed by Northern blot verification. They found that miR-29a and miR-29c levels were decreased after 7-d treatment with pioglitazone. In H9c2 cells, the effects of pioglitazone and rosiglitazone on miR-29 expression levels were blocked by a selective PPAR-γ inhibitor GW9662. Down-regulation of miR-29 by antisense inhibitor or by pioglitazone protected H9c2 cells from simulated IR injury, with increased cell survival and decreased caspase-3 activity. In contrast, overexpressing miR-29 promoted apoptosis and completely blocked the protective effect of pioglitazone. Antagomirs against miR-29a or -29c significantly reduced myocardial infarct size and apoptosis in hearts subjected to I/R-I. Western blot analyses demonstrated that Mcl-2, an anti-apoptotic Bcl-2 family member, was increased by miR-29 inhibition, similar to the finding in cancer cells. Clearly, down-regulation of miR-29 protected hearts against I/R-I through its anti-apoptotic activity. miR-29 thus represents another proapoptotic miRNA in cardiac cells, in addition to miR-1.
miRNAs and vascular apoptosis
It is known that proliferative vascular diseases share similar cellular events and molecular mechanisms with cancer, and neointimal lesion formation is the pathological basis of proliferative vascular diseases. Neointimal growth is the balance between proliferation and apoptosis of VSMCs. The increased VSMC proliferation or the relative decreased VSMC apoptosis are responsible for neointimal lesion formation. In a recent study of the potential roles of miRNAs in VSMCs proliferation and apoptosis, the authors found that miR-21 level was up-regulated in proliferative VSMCs and depletion of miR-21 resulted in decreased cell proliferation and increased cell apoptosis in a dose-dependent manner in cultured rat aortic VSMCs. This suggests that miR-21 has a proproliferative and anti-apoptotic effect on VSMCs. miR-21 inhibition upregulated, whereas miR-21 overexpression downregulated, expression of PTEN protein in VSMCs, using both “loss-of-function” and “gain-of-function” approaches. They further demonstrated that inhibition of miR-21 downregulated, whilst overexpression of miR-21 upregulated, the level of Akt protein that mediates survival signal in a cell. These results are consistent with the expression changes of PTEN. In contrast to PTEN, miR-21 knockdown decreased and overexpression increased expression of antiapoptotic Bcl-2 protein. The authors suggested that Bcl-2 might be an indirect target of miR-21 in VSMCs by suppressing expression of a gene that negatively regulates Bcl-2 expression or that miR-21 might be able to directly affect Bcl-2 expression via binding to the sequence outside the 3’UTR. Thus, Bcl-2 might be involved in the anti-apoptotic action of miR-21 in VAMCs. The authors believe that miR-21 may be a new therapeutic target for proliferative vascular diseases such as atherosclerosis, postangioplasty restenosis, transplantation arteriopathy, and stroke.
One of the major roles miRNAs play is the ability of these molecules to control cell death that bears a wide range of biological and pathological implications. While this issue has been firmly established and well advanced in the field of oncology with a realistic hope in developing novel diagnostic/prognostic biomarkers and therapeutic agents as well, relatively little is yet known about the regulation of cardiac and vascular apoptosis by miRNAs and the role of the regulation in cardiovascular pathogenesis. Nonetheless, there are many reasons to believe that miRNAs are much more importantly involved in apoptosis in the cardiovascular system than we currently know; future studies will prove it. We are now just beginning to understand their role as gatekeepers of cell death.