doi: 10.1161/CIRCULATIONAHA.108.807230
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(Circulation. 2008;118:1524-1527.)
© 2008 American Heart Association, Inc.
Editorial |
Cardiac Dissonance Without Conductors
How Dicer Depletion Provokes Chaos in the Heart
From the Department of Internal Medicine I, Junior Research Group Cardiac Wounding and Healing, Interdisciplinary Center for Clinical Research (IZKF), University Hospital, Julius Maximilians University, Würzburg, Germany.
Correspondence to Thomas Thum, MD, PhD, Medizinische Klinik und Poliklinik I, Universitätsklinikum, Julius Maximilians Universität, Josef Schneider Straße 2, D-97080, Würzburg, Germany. E-mail Thum_T{at}klinik.uni-wuerzburg.de
Key Words: Editorials • remodeling • Dicer protein • heart failure • microRNAs
Dicer was initially identified as the protein responsible for processing double-stranded RNA into small interference RNAs.1 Later, it was shown that on interaction with cofactors,2 Dicer also produces other kinds of small RNAs, ie, microRNAs (miRNAs).3 MiRNAs are endogenous small ribonucleotides that act as negative regulators of target messenger RNAs. It has been estimated that the human genome encodes up to 1000 miRNAs that modulate 30% to 50% of all genes, which demonstrates the importance of these small regulators as fundamental orchestrators of the genome.
Article p 1567
Primary transcripts (pri-miRNA) are processed in the nucleus into hairpin RNAs by the RNase III–type enzyme Drosha4 and the double-stranded RNA binding protein DGCR85 to form pre-miRNAs. On nuclear export, pre-miRNAs are processed by the ribonuclease Dicer into 19- to 25-nucleotide miRNA duplexes, of which 1 strand is implemented into the RNA-induced silencing complex, which then binds to target mRNAs with subsequent mRNA degradation or translational inhibition.6 Recent data obtained by performance of parallel transcriptome and proteomic analysis in cells transfected with different miRNAs propose that in mammals, miRNAs exert stronger effects on protein expression than on mRNA levels.7 The biological importance of Dicer-mediated maturation of miRNAs has been shown for various cell types, including embryonic stem cells8 and germline cells,9 as well as for more specialized cell types, such as pancreatic islet cells,10 immune cells,11 neural cells,12 or endothelial cells.13
A first role for Dicer in cardiac development was described previously. To assess the global requirement for miRNAs in the mouse heart early during development, Zhao and colleagues14 deleted a floxed Dicer allele using Cre recombinase under control of the endogenous Nkx2.5 regulatory region, which directs expression in cardiac progenitor cells by embryonic day (E) 8.5. This approach led to embryonic death at E12.5, with hearts displaying pericardial edema and an insufficiently developed ventricular myocardium. Interestingly, shortly before death, the authors found upregulation of genes that are activated after cardiac stress in the adult heart. Indeed, miRNA changes were also shown to trigger reexpression of a fetal gene program in the human failing heart.15 Perhaps not surprisingly, removal of the critical miRNA-processing enzyme Dicer severely disturbed spatiotemporal miRNA expression during cardiac development, with subsequent embryonic lethality. In contrast, details about the role of Dicer in the adult heart have not been reported thus far.
In the current issue of Circulation, da Costa Martins and colleagues16 used an elegant strategy to specifically investigate the cardiac effects of postnatal cardiomyocyte-specific Dicer gene loss, eg, after the initial development of the heart has been completed and a first organization of the miRNA interplay within the cardiomyocytes has been established. Using a tamoxifen-inducible Cre recombinase, the authors tested the effects of Dicer depletion in young (3 weeks old) and adult (8 weeks old) mice. Dicer loss in cardiomyocytes of young mice resulted in sudden cardiac death within 1 to 2 weeks, probably due to arrhythmias. Surprisingly, hearts displayed only a mild form of ventricular remodeling (except for dramatic atrial enlargement). In strong contrast, adult animals survived for the monitored time period but developed severe heart failure with all typical characteristics, such as ventricular enlargement, myocyte disarray, cardiac hypertrophy and fibrosis, inflammation, and interestingly, a tendency for increased capillarization (Table; Figure). When studying the miRNA expression profiles obtained from heart tissue of young and adult mice after cardiomyocyte-specific Dicer knockdown, the authors made several remarkable observations. First, the cardiac miRNA expression profiles changed significantly with age, which probably reflects alterations in maturation and differentiation processes in cardiomyocytes. Second, silencing the major miRNA processing enzyme Dicer should lead to diminished expression of miRNAs. In strong contrast to this assumption, the authors found many miRNAs to be unchanged or even upregulated in heart tissue after Dicer depletion. There are several explanations for and implications of these interesting findings. First, many miRNAs have a considerably long half-life, which may contribute to prolonged expression after Dicer depletion. In addition, because Dicer knockdown was not complete, there probably was processing of remaining miRNA by trace amounts of Dicer or by other unidentified enzymes with RNase activity. A third and most important explanation is that miRNA expression profiles were derived from all cardiac cell types despite selective knockdown of Dicer in cardiomyocytes. Thus, the relative contribution of "noncardiomyocytes" to the cardiac miRNA pool in toto dramatically increases after cardiomyocyte-selective Dicer knockdown. It is likely that miRNA depletion on Dicer knockdown leads to an initially cell-type–restricted form of cardiac stress in cardiomyocytes, the response to which is alterations of surrounding noncardiomyocytes, eg, activation of fibroblasts, endothelial cells, and various immune cells (Figure). This may also explain in part why selected miRNAs were found to have a relative increase in expression within the miRNA pool of the whole heart, which was subjected to miRNA array analysis (eg, miR-21, miR-23a/b, miR-24, and miR-805). However, the underlying molecular signals, which mediate the reactions of noncardiomyocytes after cardiomyocyte-specific Dicer depletion, remain rather unclear (eg, paracrine versus direct cell-cell interactions). Presumably, the contribution of miRNA-mediated regulation of the noncardiomyocyte fraction in the heart is of additional importance for the pathology and therapy of cardiac disease and thus should attract more attention in future research.
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After Dicer knockdown in young and adult animals, the authors found some opposing expression changes of miRNAs that were described to be specific for cardiomyocytes, such as miR-13318 and miR-208.19 For instance, miR-133a and miR-133b were strongly repressed in adult mice but not in young mice after Dicer depletion. Moreover, Chen and coworkers17 also have reported on the effects of cardiospecific Dicer knockdown by crossing mice homozygous for the floxed Dicer alleles to a transgenic mouse line in which the Cre recombinase is expressed under control of the promoter from the murine myosin heavy chain-
gene (Myh6), which resulted in cardiac-specific Dicer mutant animals. In contrast to da Costa Martins et al,16 Chen and coworkers17 found a substantial reduction of most miRNAs investigated in Dicer knockout hearts, whereas no difference was found in other tissues. All mutant mice died within 4 days after birth and displayed phenotypes of heart failure, such as sarcomere disarray and ventricular dilatation, but surprisingly, no signs of fibrosis and a reduction in heart rate19 (Table). It is surprising that Chen and coworkers17 found such a complete miRNA shutdown in the heart after cardiomyocyte-specific Dicer deletion, because many noncardiomyocytes remain unaffected by this approach and therefore should retain their miRNA expression levels. Some of the differences may be explained by variations in the technical approach (permanent versus inducible Dicer knockdown) or the chosen/given time points to investigate miRNA expression profiles (neonatal versus young versus adult animals). After induction of cardiac stress (eg, via Dicer depletion), time is required to activate or attract other cells that contribute to the noncardiomyocyte miRNA pool within the heart.20 Thus, this noncardiomyocyte miRNA pool might have been smaller in the study by Chen and colleagues.17 Furthermore, changes in MYH6 expression during cardiac development and the postnatal period (early expression at E7/8 with a subsequent decrease in the ventricle but not the atria, and another increase in expression in the ventricular tissue at E16 that lasts until the first months after birth21) may explain in part some of the differences reported in these 2 studies, because both used a genetic approach for Dicer deletion under the control of the Myh6 promoter but at different time points of cardiac development. The higher level of MYH6 expression shortly before and after birth in the atria may also contribute to the dramatic atrial enlargement after Dicer depletion in young mice.16 Future experiments, therefore, should be performed with fractionated and/or cultured pure cardiomyocytes before and after Dicer knockdown to resolve these remaining issues.
Although it is not possible to identify single miRNAs that are functionally relevant either to the observed phenotypes or to disease in general, the major accomplishment of the present study by da Costa Martins et al16 is that it provides a significant contribution to our understanding of the biological impact of miRNAs in the postnatal heart. However, new questions have arisen that must be addressed in the future. If we want to develop efficient miRNA-based therapies for cardiovascular diseases, we need to know more about the cell-type specificity of miRNAs within the heart, details concerning the timing of miRNA expression during both development and adulthood, and details about variations in the expression of targets within different cardiac cell types. We also need more information about ongoing changes in target availability, which mediates the effects of miRNA modulation within the heart. For instance, the authors observed different phenotypic effects after Dicer depletion in young versus adult animals, eg, sudden death probably due to arrhythmias in young animals but development of severe heart failure in adult animals. This might be due at least in part to the approximately 6-fold higher expression of miR-1 in hearts of young mice, because miR-1 has been shown to both directly and indirectly target several important proteins that regulate cardiac conductance.14,22,23 Obviously, over time, both miRNAs (such as miR-1) and the pool of accessible mRNA targets change, and thus, single miRNAs can exert their full effects only at certain developmental stages or under specific biological circumstances. Finally, target availability is often restricted to a given cell, which explains why miRNAs have effects in some cell types but not in others.
Three different studies have used cardiomyocyte-specific Dicer knockdown during several stages of cardiac development or postnatally with subsequent detrimental effects to the heart.14,16,17 It appears that the phenotypic consequences of Dicer depletion are time dependent, with embryonic lethality occurring after very early Dicer depletion (E8.514) and neonatal death occurring after Dicer loss at later stages during development.17 In contrast, Dicer knockdown early after birth led to death within days to weeks because of arrhythmias, whereas Dicer depletion in adult animals led to the development of cardiac failure and death within months (Table).
Alterations in Dicer function in failing hearts17 may have clinical implications. Some of the reported derailed miRNA expression patterns in the failing human heart15,24,25 may be explained by impaired Dicer function. The intriguing study by da Costa Martins and colleagues16 has provided us with important lessons about the impact of Dicer-mediated miRNA expression on disease development in the adult heart. In addition to ongoing approaches to modulate single miRNAs, treatment concepts that aim to restore cardiac Dicer and thus global miRNA processing within the failing heart could be of therapeutic relevance.
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Acknowledgments |
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Sources of Funding
This work was supported by grants from the IZKF (E-31) and the Deutsche Forschungsgemeinschaft (DFG TH903/7-1).
Disclosures
None.
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Footnotes |
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The opinions expressed in this article are not necessarily those of the editors or of the American Heart Association.
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