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Arq. Bras. Cardiol. vol.98 no.4 São Paulo Apr. 2012
Vagner Oliveira-Carvalho; Vitor Oliveira Carvalho; Miguel Morita Silva; Guilherme Veiga Guimarães; Edimar Alcides Bocchi
Instituto do Coração - InCor - HCFMUSP, SP, São Paulo, Brazil
MicroRNAs (miRNAs) are a group of newly discovered small RNAs, non-coding, which represent one of the most exciting areas of modern medical science as they modulate a huge and complex regulatory network of gene expression. Lines of evidence have recently suggested that miRNAs play a key role in the pathogenesis of heart failure. Some miRNAs highly expressed in the heart, such as miR-1, miR-133 and miR-208, are strongly associated with the development of cardiac hypertrophy, while the exact role of miR-21 in the cardiovascular system remains controversial. Serum levels of circulating miRNAs such as miR-423-5p are being evaluated as potential biomarkers in the diagnosis and prognosis of heart failure. On the other hand, the manipulation of levels of miRNAs using techniques such as mimicking the miRNAs (miRmimics) and antagonistic miRNAs (antagomiRs) is making increasingly evident the enormous potential of miRNAs as promising therapeutic strategies in heart failure.
Keywords: microRNAs / genetics; microRNAs / diagnostic use; microRNAs / antagonists & inhibitors; heart failure; cardiomegaly.
The syndrome of heart failure (HF) is considered the final common pathway of every heart disease and a major cause of death1,2. This syndrome has an alarming mortality rate of approximately 50% in five years, which can overcome many types of cancer3. In Brazil, HF represents a major cause of hospitalization for cardiovascular disease, and when considering all causes of death, it represents a mortality rate of 6.3%4,5.
The recent discovery of microRNAs (miRNAs) has placed them among the most exciting areas of modern medical science. MiRNAs are a group of small RNAs, non protein encoders, with approximately 19-25 nucleotides of extension. Differing from the wide range of RNAs encoded by the human genome, this type of RNA has been noted for its unique ability to modulate an enormous and complex regulatory network of gene expression6.
The biological role of miRNAs in the cardiovascular system of mammals has recently become a research field of rapid evolution. Several studies have demonstrated the crucial role of miRNAs not only in embryonic cardiovascular development, but also in cardiovascular disease.
Biology of microRNAs
The human genome encodes a broad range of types of RNAs, in which the function of most of these molecules has been only partially elucidated and remains unknown. Along with other more common types of RNA such as mRNA (messenger RNA or protein-coding RNA) and those with structural functions, such as tRNA (transfer RNA) and rRNA (ribosomal RNA) are the non-protein-coding RNAs, including the miRNAs.
It is known that miRNAs are usually synthesized from miRNA-specific genes or specific genetic regions that are not associated with the production of proteins (introns)7. The maturation of miRNAs involves a complex metabolic pathway that begins in the nucleus and extends to the cell cytoplasm (Fig. 1)6.
MiRNAs exert their regulatory effects by binding their nucleotides to those of the target messenger RNA (mRNA) in a process called pairing. This binding prevents the ribosomes from translating the genetic information contained in the mRNA, resulting in decreased protein synthesis of the target gene without impacting the corresponding levels of RNAm6.
The miRNA-mRNA interaction, however, does not necessarily need to be perfect, i.e., all miRNA nucleotides bound to the mRNA. In mammals, this binding is usually imperfect. Therefore, lack of necessity for a complete interaction coupled with the fact that miRNAs have small sequences, a single miRNA can regulate hundreds of target genes, and cooperate in control of a single target gene8,9.
The involvement of miRNAs in regulatory control of gene expression and association with different functions make it clear that miRNAs can alter the progression of several diseases.
MicroRNAs and the cardiovascular system
Although the biological functions of miRNAs are not fully understood, studies have shown that some miRNAs are specifically present in certain tissues or cell types, including the heart10,11. On the other hand, miRNAs whose expression is not restricted to the heart can have an important heart-specific role12. Therefore, new miRNAs are being discovered in other cells that make up the cardiovascular system, such as fibroblasts, endothelial cells and smooth muscle cells, which cannot be ignored when studying the physiology of the cardiovascular system or its response to stress.
So far, there seems to be about 150-200 miRNAs expressed in the cardiovascular system. Many of these miRNAs are dynamically regulated in response to acute cardiac stress and in some cases during long-term compensatory response of the heart to a chronic injury or hemodynamic overload13,14. Therefore, there is growing evidence that the expression of miRNAs is an important part of the mechanism of response to acute stress of the heart, contributing both to cardiac homeostasis and to the heart disease.
Some miRNAs such as miR-128, miR-302, miR-367 and miR-499 are potentially heart-specific, but more studies are needed for confirmation. Only the miRNAmiR-208 is known as heart-specific and plays an important role in the maintenance of heart development and function15,16. However, recent studies have shown that in long events such as heart injuries, this miRNA can flow into the bloodstream and be detected in peripheral blood. Thus, its levels of expression may be linked to the diagnosis and prognosis of diseases17,18.
In the skeletal muscle, mir-1, mir-133A, miR-133b and miR-206, together, account for approximately 25% of the expression of miRNAs and are often referred to as miomiRs19. The miRNAs miR-1, miR-133a and miR-133b are highly expressed in the skeletal muscle and in the heart, while the miR-206 is specifically expressed in skeletal muscle. All four muscle miRNAs are induced during muscle differentiation and play a critical role in the regulation of this process20.
MicroRNAs in cardiac hypertrophy and heart failure
Cardiac hypertrophy is also accompanied by an exchange of the genetic program that leads to the reactivation of cardiac genes normally expressed in fetal heart during embryonic development21. In 2007, a striking similarity was found between the expression pattern of miRNAs in hearts of adult individuals with heart failure and hearts of fetuses at 12-14 weeks of gestation. About 80% of miRNAs analyzed were found similarly regulated in both hearts. The most significant changes were associated with increased expression of miR-21, miR-29b, miR-129, miR-210, miR-211, miR-212, miR-423 miRNAs, and reduced expression of miR-30, miR-182 e miR-52622.
Deregulation of other miRNAs, however, has also been associated with heart failure (table 1). In an experimental mouse model in pressure overload has been applied to the heart, was one of the first changes observed was reduced expression of miR-1. This change in miR-1 expression level preceded the increase in cardiac mass and contractile dysfunction13. This result suggests that the reduction in the level of miR-1 expression may be a cause rather than an effect of the underlying pathogenesis. Therefore, both in vitro23 and in vivo24 data suggest that reduced expression of miR-1 is required for increased cell mass.
In addition to miR-1, another muscle-specific miRNA, the miR-133, also has reduced expression during cardiac hypertrophy24-26. Mice with reduced expression of miR-133 showed cardiomyopathy, heart failure and an abnormal proliferation of cardiomyocytes27. In a recent study, the expression of miR-133 was induced in a rat model subjected to acute hypertrophic stimulus. Although the weight of the heart has not been standardized, other aspects of hypertrophy, such as apoptosis and fibrosis were restored to basal levels14.
The miR-21 is one of the few miRNAs that show a regular pattern of over-expression in heart failure. Likewise, the miR-21 is also highly expressed in many cancers and cell lines, suggesting that this miRNA has a common behavior in response to stress and pathological growth of cells. However, the exact role of miR-21 in the development of cardiac hypertrophy remains controversial28.
Although the expression patterns of some miRNAs are already known and associated with heart failure, certain miRNAs may be are differentially expressed in certain types of disease. A study by Ikeda et al29 analyzed the expression patterns of miRNAs in myocardial samples of patients with ischemic cardiomyopathy, idiopathic cardiomyopathy and aortic stenosis. Interestingly, their results show that subsets of miRNAs are differentially regulated in each etiology29. Similar results were also found by Sucharov et al30. These data show that differences in expression patterns of miRNAs may be clinically important if used for the purposes of diagnosis and/or prognosis.
On the other hand, not only the subsets of miRNAs have influence on the phenotype: some specific miRNAs appear to be key regulators. In 2006, van Rooij et al26 showed that the increased expression of miR-195 in the myocardium of mice was sufficient to induce a pathological cardiac growth and heart failure within several weeks after birth. Moreover, while no phenotype was obtained by increasing expression of miR-214, miR-24 resulted in embryonic lethality. This study indicates that some specific miRNAs may play determining roles in cardiac hypertrophy26.
The drugs prescribed to the patient must also be taken into account. By using the zebrafish as a model, Simon-Sanchez et al31 demonstrated that morphine regulates the differentiation of dopaminergic neurons through the reduction of expression levels of miR-133b.31 Although the zebrafish is evolutionarily distant from man, this data indicates that the drugs may influence the expression of miRNAs.
MicroRNAs in diagnostic testing and prognosis of heart failure
Because many miRNAs are tissue specific, most clinical studies have been based on the measurement of expression levels of miRNAs in origin tissue samples. However, some miRNAs were recently found in the bloodstream and are referred to as circulating or c-miRNAs. The mechanisms involved in the release of miRNAs in the blood are not well understood. The fact that these c-miRNAs can be detected in peripheral blood make them potentially useful for fast and easy tests, assisting the diagnosis or guiding therapy.
The first study in mice showed that the plasma level of miR-208 (heart-specific miRNA) is related to myocardial injury and is detectable after induction of this injury18. In humans, the miRNAs miR-132,33, miR-13334, miR-208a35 and miR-49936 have been proposed as good biomarkers of acute myocardial infarction, showing significantly higher plasma levels compared to patients without this condition.
Cheng et al32 reported that the profiles of miRNAs are differentially expressed in the myocardium based on the etiology of heart failure, suggesting that each form of etiology is characterized by a distinct expression profile of miRNA32. However, the need for an invasive procedure to obtain samples from the myocardium makes the clinical application of this approach very limited. However, recent evidence has shown that the c-miRNA miR-423-5p shows an increased expression during heart failure and can be used as a biomarker37.
In 2009, Matkovich et al. evaluated the expression profile of miRNAs in heart failure patients before and after treatment with left ventricular assistance devices. Interestingly, 71.4% of miRNAs differentially regulated in heart failure were normalized after treatment38. These results suggest that miRNAs may serve as markers of myocardial recovery in patients with advanced heart failure.
MicroRNAs in the treatment of heart failure
Two therapeutic strategies involving the knowledge of miRNAs have been recently studied: the use of antagomirs and miR-mimics. These strategies are based on the normalization of tissue level of specific miRNAs, silencing those who have over-expressed or replacing those that have a deficit in expression in pathological processes (Fig. 2).
In a pathological condition in which certain miRNAs are over expressed, the first thing one think is how to intervene in the effect caused by the excessive increase in the expression of these miRNAs. For this purpose, a class of antimiRNAs called antagomirs was developed.
Antagomirs are small antagonistic nucleotide sequences, of single strands, artificially synthesized to be perfectly complementary to a specific mature miRNA. When injected systemically or locally, antagomirs interact with miRNAs in the cytoplasm and hybridize specifically to the target mature miRNA making it difficult to bind the miRNA with its respective mRNA. Thus, the antagomirs act as competitive inhibitors of miRNA and lead to a decrease in the effect caused by excessive increase in the expression of certain miRNAs39.
Far from being utopian, this therapeutic strategy has already been studied by many researchers. In a pioneering study, Thum et al40 induced mice to cardiac hypertrophy through pressure overload. After three weeks, the mice received antagomir designed to functionally inhibit miR-21 (miRNAs over-expressed in cardiac fibroblasts during hypertrophy). As a result, it was observed that the mice showed a significant regression of cardiac hypertrophy and fibrosis, as well as attenuation of impairment of cardiac function40. Another successful approach was published in 2011 by Montgomery et al41. in which the antimiR-208a was systemically administered during hypertension induced by heart failure in hypertensive rats leading to a potent silencing of miR-208a in the heart. The therapeutic inhibition of miR-208a avoided the pathological change of myosin and cardiac remodeling, improving cardiac function and survival41.
These results demonstrate that the use of antagomirs can be useful in preventing and/or reversing cardiac hypertrophy. However, most studies to date have focused on "silencing" only isolated miRNAs. However, considering that more than one miRNA may be involved in the pathological process, probably several miRNAs must be silenced to obtain an effective therapy.
Just as the increased expression of some miRNAs may be related to the outbreak of pathogenic processes, decreased expression of specific miRNAs can also lead to a pathological state.
The intervention to be made to normalize the level of expression of these miRNAs, however, is based on the administration of molecules that will functionally mimic natural miRNAs.
The miRmimics are short double-strand artificial nucleotide sequences that resemble miRNA precursors (pre-miRNA). When introduced into cells, the miR-mimics are recognized by the miRNA biogenesis machinery and processed by the enzyme Dicer, and then incorporated into the RISC enzyme complex. Thus, the mimics will work as a replacement of little expressed miRNAs by regulating the target mRNA as endogenous miRNAs42.
The replacement of miRNAs, however, is subject to an additional obstacle: specificity. The miR-mimics should act only on the target tissue. Otherwise, as if administered systemically, they could result in one or more miRNAs exercising regulatory function in tissues where these miRNAs are not normally expressed. This erroneous regulation would likely lead to triggering side effects.
To overcome this obstacle, more complex and accurate management systems are required. To this end, the use of viral vectors has proven to be promising. These vectors are produced by bioengineering from non-pathogenic viruses belonging to the Parvovirida family and have a high affinity with the myocardium43.
Like antagomirs, the therapeutic effectiveness of miR-mimics is also being studied. In a study by Suckau et al., a viral vector optimized with mimics has been successfully used in rats with pressure overload. As a result, the authors observed that there was a normalization of cardiac dilation and a significant reduction of cardiac hypertrophy, cardiomyocyte diameter and cardiac fibrosis44.
Understanding the biology of miRNAs and their role in pathogenic processes is an exciting new milestone in cardiovascular medicine. The potential of miRNAs is increasingly evident as new tools in the diagnosis and prognosis, as well as promising therapeutic strategies in many sub-areas of cardiology, including heart failure. However, before becoming a reality, many studies are still needed. Overcoming obstacles, miRNA-based therapies can become part of the arsenal of cardiologists in the treatment, diagnosis and prognosis of heart failure.
Potential Conflict of Interest
No potential conflict of interest relevant to this article was reported.
Sources of Funding
There were no external funding sources for this study.
This study is not associated with any post-graduation program.
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Mailing Address: Manuscript received August 31, 2011; revised manuscript received October 25, 2011; accepted November 10, 2011.
Vagner Oliveira Carvalho
Av. Dr. Enéas de Carvalho Aguiar, 44
Laboratório de Insuficiência Cardíaca e Transplante - InCor - Bloco 1, 1º Andar
05403-900, São Paulo, SP, Brazil
Manuscript received August 31, 2011; revised manuscript received October 25, 2011; accepted November 10, 2011.