Published online before print February 23, 2009, doi: 10.1161/CIRCULATIONAHA.108.813576
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(Circulation. 2009;119:1263-1271.)
© 2009 American Heart Association, Inc.
Molecular Cardiology |
Reciprocal Regulation of Myocardial microRNAs and Messenger RNA in Human Cardiomyopathy and Reversal of the microRNA Signature by Biomechanical Support
From the Center for Pharmacogenomics, Washington University, St Louis, Mo (S.J.M., D.J.V.B., A.D., W.H.E., L.E.D., G.W.D.); Department of Cardiology, Methodist Hospital and Methodist DeBakey Heart Center, Houston, Tex (K.A.Y., G.T.-A.); Department of Pathology and Immunology, Washington University, St Louis, Mo (M.A.W.); and Cardiovascular Research Institute, University of Pennsylvania School of Medicine, Philadelphia (K.B.M.).
Correspondence to Gerald W. Dorn II, MD, Washington University in St Louis, Center for Pharmacogenomics, 660 S Euclid Ave, Campus Box 8086, St Louis, MO 63110. E-mail gdorn{at}dom.wustl.edu
Received August 8, 2008; accepted December 19, 2008.
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Abstract |
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Background— Much has been learned about transcriptional control of cardiac gene expression in clinical and experimental congestive heart failure (CHF), but less is known about dynamic regulation of microRNAs (miRs) in CHF and during CHF treatment. We performed comprehensive microarray profiling of miRs and messenger RNAs (mRNAs) in myocardial specimens from human CHF with (n=10) or without (n=17) biomechanical support from left ventricular assist devices in comparison to nonfailing hearts (n=11).
Methods and Results— Twenty-eight miRs were upregulated >2.0-fold (P<0.001) in CHF, with nearly complete normalization of the heart failure miR signature by left ventricular assist device treatment. In contrast, of 444 mRNAs that were altered by >1.3-fold in failing hearts, only 29 mRNAs normalized by as much as 25% in post–left ventricular assist device hearts. Unsupervised hierarchical clustering of upregulated miRs and mRNAs with nearest centroid analysis and leave-1-out cross-validation revealed that combining the miR and mRNA signatures increased the ability of RNA profiling to serve as a clinical biomarker of diagnostic group and functional class.
Conclusions— These results show that miRs are more sensitive than mRNAs to the acute functional status of end-stage heart failure, consistent with important functions for regulated miRs in the myocardial response to stress. Combined miR and mRNA profiling may have superior potential as a diagnostic and prognostic test in end-stage cardiomyopathy.
Key Words: cardiomyopathy • diagnosis • genes • heart-assist device • microRNA
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Introduction |
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One of the promises of transcriptional profiling is that RNA patterns from diseased tissues will enhance the accuracy of clinical diagnosis and prognostication. To date, this approach has been most successful for cancer, in which characteristic transcriptional signatures of numerous malignancies have provided insight into causation1,2 and outcome.3–5 A similarly pressing need exists for novel molecular diagnostics to better assess heart failure to direct optimal management.
Clinical Perspective p 1271
Previous messenger RNA (mRNA) profiling studies in heart failure revealed that distinct mRNA signatures detected early in the course of the disease can differentiate between cardiomyopathies of ischemic and nonischemic causes and provide prognostic information.6,7 In contrast, mRNA signatures of end-stage cardiomyopathy vary little between different causes and do not reflect striking improvements in functional performance provided by biomechanical support.8–13 These findings suggest that additional potent factors are regulating the interaction between transcript abundance and tissue phenotype.
A hallmark of heart failure mRNA signatures is that more transcripts are downregulated than are upregulated,14,15 suggesting the importance of molecular mechanisms that suppress mRNA steady state levels. MicroRNAs (miRs) are small, noncoding RNAs that bind mRNAs at their 3' untranslated regions, stimulating mRNA degradation or inhibiting protein translation.16 Many miRs are upregulated in response to cellular stress17 and can modify essential cellular functions of proliferation, differentiation, and programmed death.18–20 miR signatures are being used as markers of cancer pathogenesis21,22 and to predict cancer course.23–25 A recent explosion of experimental data indicates that miRs are also regulated in cardiac disease26–28 and have the capacity to create cardiac pathology.29–31 Given that miRs respond to acute changes in cell stress, we hypothesized that combining information from myocardial miR profiles and mRNA signatures could reconcile discrepancies between mRNA levels and cardiac phenotype during heart failure and reverse remodeling. We examined this notion through comprehensive analyses of miR and mRNA expression levels in myocardial samples from patients with end-stage heart failure, off and on biomechanical support with left ventricular assist devices (LVADs), in comparison with normal heart samples.
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Methods |
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Tissue Cohort
Nonfailing and failing tissues were obtained from the University of Pennsylvania Cardiovascular Research Institute, Philadelphia, and LVAD-treated tissues (average time, 1.7 months) were obtained from the Methodist Hospital, Houston, Tex, in accordance with institutional review board–approved protocols. RNA integrity and quality were assessed before microarray analysis by gel electrophoresis and on an Agilent Bioanalyzer and showed no differences in pass/fail rate of samples obtained from different centers. Clinical parameters for all subjects are described in Table I in the online-only Data Supplement. All samples were subjected to miRNA profiling. RNA amounts limited mRNA array analysis to 6 nonfailing, 13 failing (4 ischemic, 9 nonischemic), and 4 post-LVAD (3 ischemic, 1 nonischemic) samples.
Microarrays
Total RNA was isolated with the use of Trizol reagent (Invitrogen, Carlsbad, Calif). MicroRNA hybridization was performed by Invitrogen Custom Services with the use of the NCode Multi-Species miRNA Microarray version 2, which recognizes 467 separate human miRs in accordance with the Sanger Institute miRBase version 9.0.32 Data were analyzed with the use of Invitrogen NCode algorithms. miR nomenclature in this article reflects miRBase version 11.0, April 2008. Total RNA from the same specimens was processed and hybridized to Affymetrix HuEx version 1.0 arrays by the Multiplexed Gene Analysis core at Washington University. A total of 232 448 probe sets corresponding to well-annotated and confirmed exons are represented, grouped into 21 980 transcript clusters (whole genes) by Partek Genomics Suite version 6.3 software (Partek, St Louis, Mo) with the use of Affymetrix metafiles. Expression data were analyzed with Partek. All miR and mRNA array data, regardless of tissue origin, were imported and normalized together. Significance (probability values) of miR expression changes was determined with a bootstrapping algorithm from the distribution of residuals used in the initial data-fitting model (see Methods in the online-only Data Supplement). Partek software was used to compute significance of mRNA expression changes with 1-way ANOVA at a false discovery rate of 0.03. Pairwise comparisons were significant at P=0.001. Detailed analytical methods are provided in Methods in the online-only Data Supplement.
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
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Results |
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miR Expression Profile of End-Stage and LVAD-Supported Hearts
Forty-eight myocardial samples were obtained. Unamplified RNA was prepared from each and was suitable in quantity and quality for multiplatform analysis in 38 (11 nonfailing, 17 end-stage failing, and 10 LVAD-supported). Characteristics of the study subjects are in Table I in the online-only Data Supplement.
Myocardial miR profiles were examined with Invitrogen NCode Multi-Species miRNA Microarray version 2, on which triplicate probes for 467 separate human miRs are represented. After signals below the threshold level for detection were filtered, 81 miRs, including 25 of the 34 (74%) listed in miRBase 8.2 as being specific for human cardiovascular tissue,33 were confidently detected (see Methods in the online-only Data Supplement for threshold determination) in 1 or more of the 3 clinical groups (Table II in the online-only Data Supplement).
Twenty-eight confidently detected miRs were significantly upregulated in cardiomyopathic hearts (n=17), defined as a 
2.0-fold increase over nonfailing hearts (n=11; P<0.001) (Figure 1, top, and Table III in the online-only Data Supplement) and designated as "heart failure" miRs. Three miRs previously reported to be regulated in experimental or human cardiac disease, miR-133a, miR-21, and miR-23a,26,31 showed strong trends for being increased at probability values between 0.001 and 0.01 (Figure 1, top, and Table III in the online-only Data Supplement). No miRs met the same level of stringency (P<0.001; 50% decrease) for downregulation in heart failure.
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Biomechanical support with LVADs can produce striking improvements in contractile performance and reverse ventricular remodeling in end-stage cardiomyopathy.9–11,13 However, global myocardial mRNA expression signatures do not reflect clinical improvements in post-LVAD hearts.8 We examined miR expression levels in myocardium from LVAD-supported hearts (n=10). The overall trend was for reversal or normalization of the heart failure miR signature (Figure 1, top, and Table III in the online-only Data Supplement). Twenty of the 28 heart failure miRs showed full normalization or significant decreases in expression level between the cardiomyopathy and LVAD-treated groups (Tables IIIa and IIIb in the online-only Data Supplement). The remaining 8 heart failure miRs showed intermediate values that were not significantly different from cardiomyopathy (Table IIIc in the online-only Data Supplement). In no instance did a miR that was not primarily regulated in heart failure show altered expression in LVAD-supported myocardium.
Unsupervised clustering of the expression profiles for cardiac miRs in cardiomyopathy and nonfailing hearts revealed a distinct heart failure miR profile (Figure 1, bottom, and Figure I in the online-only Data Supplement). Eight of the 10 LVAD samples were grouped between the nonfailing and failing clusters but were not recognized as a distinct class (Figure 1, bottom). Within this group, subclustering based on the branching points of the dendrogram identified 3 samples with a less normal profile than the others (P4, P7, P10) and a fourth sample that was clearly abnormal (P3) (Figure 1, bottom), but no clinical feature was found that clearly distinguished these 4 samples from the other LVAD-treated hearts.
Nearest centroid analysis with leave-1-out cross-validation, with the signature of 28 heart failure miRs, correctly classified 64% of nonfailing samples, 94% of failing samples, and 50% of post-LVAD samples. A single failing sample was incorrectly assigned to the nonfailing group, and 2 of the 4 misclassified nonfailing hearts (NF3 and NF4) had cardiac masses >500 g, consistent with substantial overlap between miR profiles for heart failure and cardiac hypertrophy.26 Three of the 10 post-LVAD samples were designated as nonfailing and 2 as failing, raising the possibility that myocardial miR levels are particularly sensitive to functional status.
mRNA Expression Profiles of Heart Failure and Heart Failure Recovery
We used Affymetrix HuEx version 1.0 arrays to perform a comprehensive analysis of mRNA expression from a subset of the hearts on which we had miR expression profiles, excluding the hypertrophied, nonfailing hearts (6 normal, 13 heart failure, 4 post-LVAD). Of 21 980 core genes represented on the array, 13 382 genes were confidently detected in the pooled set of samples (see Methods in the online-only Data Supplement for threshold determination). Comparative analysis of nonfailing and failing mRNA profiles showed significant regulation (defined as >1.3-fold change; P<0.001; false discovery rate 0.03) of 444 genes in heart failure, of which 155 (35%) were upregulated and 289 (65%) were downregulated (Table IV in the online-only Data Supplement). The microarray gave similar results for the signature regulated "fetal genes," as have prior studies of mRNA expression in human heart failure (Table V in the online-only Data Supplement). Functional classification of mRNA transcripts by Gene Ontology (http://www.geneontology.org) terms showed a preponderance of dysregulated genes associated with receptor signaling, cytoskeletal, transcriptional, and metabolic processes (not shown).
Prior studies in which older analytical platforms were used to compare cardiac mRNA expression before and after LVAD implantation identified a limited number of dysregulated transcripts that corrected with LVAD treatment.8,34–36 In our samples, only 12 of the 155 upregulated mRNAs (7.7%) and 17 of the 290 downregulated mRNAs (5.9%) normalized by at least 25% (Figure 2, top, and Table 1). Four of the 17 downregulated genes (ANKRD2, C18orf1, GADD45B, MT1H) and 2 of the 13 upregulated genes (CRYM, SLC14A1) were previously shown to recover with LVAD treatment.8,34 Hierarchical clustering of the expression profiles for the 50 most upregulated cardiac mRNAs across the cardiomyopathy and nonfailing samples revealed distinct transcriptional profiles for normal and failing hearts (Figure II in the online-only Data Supplement). However, when the 4 LVAD-treated mRNA profiles were added to the analysis, they scattered within the cardiomyopathy grouping (Figure 2, bottom). Nearest centroid analysis with the use of the 50 most upregulated mRNAs as variables correctly classified 100% of nonfailing samples and 77% of failing samples but only 50% of post-LVAD samples. Each of the misclassified failing samples were assigned to the post-LVAD group, and half of the post-LVAD samples were incorrectly classified as failing, consistent with the established tendency of most dysregulated mRNAs not to normalize in LVAD-supported hearts.8
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Combined miR and mRNA Profiles as a Molecular Signature for Cardiac Failure and Recovery
In the aforementioned studies, the mRNA signature was better at differentiating between nonfailing and cardiomyopathic hearts but failed to distinguish between failing and recovering (LVAD-supported) myocardium. In contrast, the miR signature was exquisitely sensitive to the mechanical unloading status of end-stage hearts but failed to differentiate some hearts subjected to biomechanical unloading from those in the nonfailing group. We tested whether an analysis combining upregulated RNAs from both signatures increased the utility of RNA profiling as a biomarker. Combining the miR and mRNA profiles resolved each of the misidentified samples and clustered the LVAD-supported population as a near-normal subset of cardiomyopathic hearts (Figure 3). Nearest centroid analysis of the combined miR and mRNA signatures correctly classified 100% of nonfailing and 100% of post-LVAD samples and 92% of failing samples (1 failing sample was incorrectly assigned to the post-LVAD class).
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Discussion |
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In a comprehensive examination of miR and mRNA expression from failing, nonfailing, and LVAD-supported human myocardium, we show that the cardiac miR signature is an exquisitely discriminating biomarker of the severely failing heart and that its response to biomechanical unloading and greatly enhances the predictive ability of mRNA profiles to categorize the clinical status of heart failure.
The miRNA Signature of Human Cardiomyopathy
Only a handful of previous studies have examined miR levels in failing human hearts, and the results are not consistent.26,28,31,37,38 Here, using stringent criteria, we identified 
30 miRs that are upregulated in failing human myocardium. These findings confirm 4 of 5 miRs initially reported in the seminal myocardial miR study26 to be upregulated in failing human myocardium by Northern blot analysis (miR-24, miR-125b, and miR-195). In addition, our results suggest that miR-21, miR-23a, and miR-199a-3p, which in that study and a subsequent report were increased in murine heart disease,26,39 are also regulated in the human condition. Notwithstanding some variance attributable to diverse study cohorts and different analytical platforms, the 3 larger miR studies of human heart failure to date (ie, studies of 6 diseased hearts; present study plus References 37 and 38) demonstrate consistent upregulation of miR-23a, miR-125b, miR-195, and mir-199a-3p, and a plurality agree on upregulation of miR-24, miR-27 (present study and Reference 37), and miR-26b (present study and Reference 38) (Table 2). We believe that the available data are sufficient to confidently assert that these 8 miRs are part of a "heart failure" miR program. miR-1, which is reported to have proarrhythmic consequences,29,40 is upregulated in subjects with coronary artery disease40 in the present study and in 1 other heart failure study28 but in other heart failure surveys has been either invariant or downregulated.37,38 It is possible that assays of this muscle-specific miR are especially sensitive to fibrous content of the myocardial sample.
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The most striking finding of the end-stage heart failure miR signature is the extent to which it is normalized by LVAD support. This distinguishes it from the mRNA signature of end-stage cardiomyopathy, which is largely invariant. Although specific mRNAs appear to reflect functional changes in the heart after β-blockade41 or left ventricular synchronization therapy,42 the vast majority of regulated cardiac genes do not mirror functional improvements.8 Our results suggest that miRs are more reactive than mRNAs to acute alterations in pathophysiological status, which may make them better molecular markers of stress and the stress response. It will be of interest to determine whether the broad normalization of miR profile we observed with LVAD therapy is reproduced by pharmacological treatment with β-blockers and angiotensin-converting enzyme inhibitors or with ventricular resynchronization.
Several of the heart failure–regulated miRs in this study have been implicated in relevant cellular signaling pathways. miR-24, -26, -27, and -103 are induced in response to a hypoxic cellular microenvironment.43 Expression of let-7f and miR-27b favors angiogenesis,44 whereas increased levels of miR-29b promote apoptosis via downregulation of Mcl-1.45 Increased expression of miR-199a-3p may be proapoptotic by virtue of its ability to downregulate the survival kinase ERK2.46 Overexpression of miR-195 and -199a in cardiomyocytes provoked myocyte enlargement, whereas overexpression of miR-195 in the mouse heart caused myocyte hypertrophy that progressed to dilated cardiomyopathy.26 As genetic models of miR upregulation are developed, a more complete understanding will be developed of the role of other regulated miRs, alone and in combination, on interdiction of mRNA translation and destabilization of mRNA targets in heart disease.
Clinical Utility of miR and mRNA Profiling
Transcriptional profiling performed at multiple centers has revealed a characteristic pattern of cardiac gene regulation in cardiomyopathy. Whereas the myocardium of early heart failure retains sufficient molecular plasticity for mRNA signatures to vary with cause and prognosis,6,7,47 the mRNA signature of end-stage cardiomyopathy appears to be relatively invariant and has not provided insight that adds to standard clinical metrics.8
Several miRs have been identified that are regulated in cardiac hypertrophy and heart failure and can participate in cardiac remodeling,26 but whether these miRs were dynamically regulated in failing versus recovering end-stage cardiomyopathy was unknown. As an initial step toward deriving a useful clinical biomarker for heart failure recovery, the present results clearly demonstrate synergism between miR and mRNA profiling in assessing the functional status of end-stage cardiomyopathic hearts and show that miRs are more sensitive than mRNAs to the functional status of end-stage heart failure. These findings support important functions for regulated miRs in the acute myocardial response to stress. Given this increased sensitivity, it is tempting to speculate that miRs may also prove useful as diagnostic and prognostic markers in cardiomyopathies of less severity and shorter duration. Such application is consistent with, and complementary to, recent consensus statements48 on the clinical use of endomyocardial biopsy in cardiomyopathy and heart failure.
Study Limitations
We applied a very high level of statistical rigor in the studies, accepting only results that were both statistically significant (P<0.001) and unambiguously regulated (fold-expression >2.0 for miR and >1.3 for mRNA). It is certain that we fail to report miR and mRNA changes, including some of substantial biological significance, that occur in heart failure. Indeed, the fact that we did not identify significantly downregulated miRs in the present study should not be interpreted as suggesting that downregulation does not occur, but rather that for the purposes of developing a clinical tool we opted for specificity, rather than sensitivity, of the raw findings. Furthermore, we did not have access to pre- and post-LVAD cardiac samples from the same individuals. The concordance of our miR and mRNA results with previous studies suggests that the dynamic miR regulation we observe is not a result of sampling error but reflects true differences between cardiac miR and mRNA regulation. Finally, it is possible that normalization of both miR and mRNA profiles occurs after LVAD therapy but over different periods of time. On the basis of the absence of mRNA normalization in a previous, larger study,8 we believe that this is unlikely. However, serial studies before and after biomechanical support in the same individual will be an important next step to establishing the utility of combined miR and mRNA profiling in clinical assessment of end-stage heart failure.
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Acknowledgments |
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Sources of Funding
This publication was made possible by National Institutes of Health grants HL59888, 77101, 80008, and 87871 to Dr Dorn and R01 AG017022 to Dr Margulies and by support from UL1 RR024992 from the National Center for Research Resources.
Disclosures
None.
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The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.108.813576/DC1.
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Circulation 2009 119: 1177-1179.
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