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 (nϭ10) or without (nϭ17) biomechanical support from left ventricular assist devices in comparison to nonfailing hearts (nϭ11). Methods and Results-Twenty-eight miRs were upregulated Ͼ2.0-fold (PϽ0.001) 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 Ͼ1.3-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: RNA sequencing Ⅲ microarray Ⅲ gene regulation Ⅲ Gq S ignaling factors that mediate cardiac hypertrophy and heart failure do so in large part by altering gene expression. Accordingly, physiological hypertrophy, pathological hypertrophy and heart failure are associated with distinct transcriptional signatures in human disease and experimental mouse models. 1,2 The ability to detect early changes in myocardial gene expression is essential to understanding pathophysiological mechanisms in experimental models, and is predicted to provide crucial diagnostic and prognostic information in human heart disease. 3 We developed techniques for broad, accurate, and inexpensive characterization of individual mouse cardiac transcriptional signatures using massively parallel resequencing of heart cDNA libraries using the Illumina Genome Analyzer II. The digital readouts enable parallel quantification and annotation of myocardial transcripts, including mRNAs expressed at levels below 1 copy per cell. Here, we describe the components of a molecular and bioinformatic "RNA sequencing pipeline" optimized for comparative evaluation of transcript signatures in genetic mouse heart models. We compare transcriptional profiling by Illumina mRNA sequencing with Affymetrix microarray RNA in mouse hearts with a well characterized transcriptional signature of pathological hypertrophy, the G␣q transgenic mouse, 4,5 to illustrate the advantages of RNA sequencing over array-based platforms. MethodsAn expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org. Mouse ModelsGeneration of the G␣q-40 transgenic mouse line has been described previously. 4,5 Four pairs of 8-week-old male nontransgenic FVB/N and G␣q-40 transgenic mouse hearts were used for RNA sequencing studies. Echocardiographic and cardiac catheterization studies were performed using standard methods. 4,6 Preparation and Quantification of Total Myocardial RNA Total RNA was isolated from flash-frozen mouse hearts using TRIzol (Invitrogen) according to the directions of the manufacturer, except that isopropyl alcohol precipitation of RNA was allowed to proceed for 30 minutes at room temperature. RNA was quantified via NanoDrop or UV spectrometer and integrity (28S:18S ratio) was assessed on 1% agarose.

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Sporadic heart failure is thought to have a genetic component, but the contributing genetic events are poorly defined. Here, we used ultra-high-throughput resequencing of pooled DNAs to identify SNPs in 4 biologically relevant cardiac signaling genes, and then examined the association between allelic variants and incidence of sporadic heart failure in 2 large Caucasian populations. Resequencing of DNA pools, each containing DNA from approximately 100 individuals, was rapid, accurate, and highly sensitive for identifying common and rare SNPs; it also had striking advantages in time and cost efficiencies over individual resequencing using conventional Sanger methods. In 2,606 individuals examined, we identified a total of 129 separate SNPs in the 4 cardiac signaling genes, including 23 nonsynonymous SNPs that we believe to be novel. Comparison of allele frequencies between 625 Caucasian nonaffected controls and 1,117 Caucasian individuals with systolic heart failure revealed 12 SNPs in the cardiovascular heat shock protein gene HSPB7 with greater proportional representation in the systolic heart failure group; all 12 SNPs were confirmed in an independent replication study. These SNPs were found to be in tight linkage disequilibrium, likely reflecting a single genetic event, but none altered amino acid sequence. These results establish the power and applicability of pooled resequencing for comparative SNP association analysis of target subgenomes in large populations and identify an association between multiple HSPB7 polymorphisms and heart failure.

Epigenetic processes have been implicated in the pathophysiology of alcohol dependence, but the specific molecular mechanisms mediating dependence-induced neuroadaptations remain largely unknown. Here, we found that a history of alcohol dependence persistently decreased the expression of Prdm2, a histone methyltransferase that monomethylates histone 3 at the lysine 9 residue (H3K9me1), in the rat dorsomedial prefrontal cortex (dmPFC). Downregulation of Prdm2 was associated with decreased H3K9me1, supporting that changes in Prdm2 mRNA levels affected its activity. Chromatin immunoprecipitation followed by massively parallel DNA sequencing showed that genes involved in synaptic communication are epigenetically regulated by H3K9me1 in dependent rats. In non-dependent rats, viral-vector-mediated knockdown of Prdm2 in the dmPFC resulted in expression changes similar to those observed following a history of alcohol dependence. Prdm2 knockdown resulted in increased alcohol self-administration, increased aversion-resistant alcohol intake and enhanced stress-induced relapse to alcohol seeking, a phenocopy of postdependent rats. Collectively, these results identify a novel epigenetic mechanism that contributes to the development of alcohol-seeking behavior following a history of dependence.